Biochem Lec Merged

BIOCHEMISTRY FIRST SEMESTER
LECTURE | CHRISTIAN JOHN CAPIRIG, MD | MIDTERM A.Y. 2021 - 2022

CARBOHYDRATES AND GLYCOBIOLOGY
OUTLINE
I. Sugars: Structures and Stereochemistry
II. Reactions of Monosaccharides
III. Important Oligosaccharides
IV. Structures and Functions of Polysaccharides
V. Glycoconjugates: Proteoglycans, Glycoproteins,
and Glycolipids
CARBOHYDRATES
• In scientific literature, sugar is also known as
carbohydrate (or hydrate of carbon)
• Most abundant biomolecules on Earth • Monosaccharides: monomers (building blocks) of all
o Each photosynthesis converts more than 100 carbohydrates.
billion times of carbon dioxide and water into • Called differently depending on the number of
carbohydrates. Certain carbohydrates are a carbons and main functional group.
dietary staple of most parts of the world. Rice is • Simplest monosaccharides contain three carbons
covered with starch, which is a form of (triose).
carbohydrates. The oxidation of carbohydrates is o Even if they have a ketone or aldehyde in their
a central energy yielding pathway in most non- functional group, as long as, they have three
photosynthetic cells. We consume carbons, we call them as triose.
carbohydrates to yield energy. And if we don’t use o Glyceraldehyde (aldotriose)
it, at the moment, we store them in another form. ▪ Simplest aldotriose
o Insoluble carbohydrate polymers serve as ▪ Aldehyde as functional group, followed by a
structural and protective elements in the cell wall hydrogen, then hydroxyl group on the second
of bacteria and plants and in the connective tissue carbon and another hydroxyl group on the
of animals. Other carbohydrates lubricate skeletal third carbon. There is multiple hydroxides,
joints and participate in recognition and adhesion hydroxyl groups. Hence, it is poly OL.
between cells. Most complex carbohydrate o Dihydroxyacetone (ketotriose)
polymers are covalently attach the proteins or ▪ The simplest ketotriose
lipids and act as signals. ▪ Hyroxyacetone has its functional group on
• Has three major size classes: monosaccharides, the second carbon. The same structure will
oligosaccharides, and polysaccharides come up even if you reverse or rotate it on
• Polyhydroxyaldehydes (aldose) or different direction.
polyhydroxyketones (ketose)
• Name ends in -ose
• Many, but not all, have the empirical formula: (CH2O)n
o n = number of molecules
o The empirical formula for carbohydrates contains
of C and a molecule. The water is known as
hydrate, hydrating the carbon.
o Some contain nitrogen, phosphorous, or sulfur
• Play a number of important roles in biochemical
processes • Monosaccharides also exhibit stereoisomerism
o Major sources of energy o Stereoisomerism is the difference in the
▪ Go foods to spend energy in doing activities configuration of a chemical structure in space.
o Have a key role in cell surfaces such as cell-cell Even if their connectivity is the same but their
interactions and immune recognition orientation in space is different—they would be
o Essential structural components different compounds.
WHAT IS UNIQUE ABOUT THE STRUCTURE OF o Simplest carbohydrate that exhibits
SUGARS? stereoisomerism is glyceraldehyde.
Dihydroxyacetone has its functional group in the
TELETUBBIES AND FRIENDS 1

middle of its structure. So, rotating or reversing it
would yield the same structure.
• Mirror-image stereoisomers are called enantiomers.
• Non mirror-image stereoisomers are called
diastereomers
• Configuration: three-dimensional arrangement of
groups around the chiral carbon.
o Chiral carbon contains four different groups
attached to it. Stereoisomers differ from each
If you are determining if it is L or D, you would always
other in configuration.
look at its second to the last carbon.
• Stereoisomers differ from each other in configuration.

o D,L system is widely used This is a wedge bond, meaning directed towards the
o R,S system is also used, a more recent system back (vertical). The same thing for the solid bond,
▪ No one-to-one correspondence between the projecting towards you (horizontal).
two systems (e.g., some D-isomers are R,
while others are S)
• Possibilities for stereoisomerism increase as the
number of carbon atoms increases.
o The number of stereoisomers for four carbon
monosaccharides increases.
• To show structures of the molecules, Fischer
projection is used.
o Fischer was developed by Emil Fischer during
1970s or 1980s.
o Shows the two-dimensional perspective of the
molecular structure
▪ It is the best projection for liner
carbohydrates. We would be able to
distinguish if it is a galactose or whatever that
monosaccharide is. We would be able to
know if it is a levo-rotatory or dextro-rotatory
molecule.
• In a Fischer projection, the orientation of the bonds Numbering of carbon atoms in sugars. (a) Examples of
represent the direction of the bond an aldose (D-glucose) and a ketose (D-fructose), showing
o Vertical bonds: directed behind the plane the numbering of carbon atoms. (b) A comparison of the
▪ Towards the back; away from the viewer structures of D-glucose and L-glucose.
o Horizontal bonds: directed in front of the plane • This is how we number carbon atoms in sugars.
▪ Towards the viewer Showing on the picture above is an aldose and
ketose.
• At A. The one involved in the aldehyde group is
written at the top—the most oxidized because of its
double bond, designated as the carbon one. In the

ketose shown, the ketone group becomes C2—the same configuration. However, they have different
carbon atom, next to the top. Note: the carbon configurations and arrangements. Both D-Erythrose
containing the priority functional group is closest to and D-Threose have OHs on the plane’s right side.
the number one carbon. The designation of the However, for the Erythrose second carbon, the OH is
configuration if it is L or D, depending on the chiral found on the right but for D3, it is on the left. If you try
carbon’s arrangement with the highest number. In the to superimpose the D-Threose and D-Erythrose with
case of both glucose and fructose, this is carbon 5, each other, they are not mirror images of each
containing six carbons. To decide whether it is an L or other—non stereoisomers are called
D configuration, it will depend on the carbon with the diastereomers. So, D-Erythrose and L-Erythrose are
highest number (highly oxidized). It should always be enantiomers whereas, the D-erythrose and L-
the penultimate carbon or carbon which is second Threose are diastereomers. Diastereomers that
to the last of the numbering system. differ from each other in the configuration having only
• At B, they are the mirror of each other. So they are one chiral carbon are called epimers. If it is multiple
enantiomers. carbons are no longer called an epimer so it is
generally diastereomer.
• Most of the carbohydrates that exist in the body are
aldohexoses, so we do not talk about ketotetroses.
They are not that abundant except for sugar, such as
sucrose where it’s a dimer of glucose and a fructose
(ketohexose). Aldopentoses have three chiral
carbons. Aldotetroses have three chiral carbons.
Aldopentoses have three chiral carbons.
Aldohexoses have four chiral carbons. Aldopentoses
have two to the third power—possibility for 8
isomers (4D and 4L).
• Most of the sugars, we encounter in nature, especially
in food containing either five or six carbon atoms.
Glucose is the go-to carbohydrate when it comes to
energy-yielding. Glucose is an ubiquitous energy
source and ribose plays an important role in the
structure of nucleic acids.
This is what happens as another carbon is added to

glyceraldehyde to give carbon for carbon sugar.
• Observe A (upper portion). You add carbon sugar on
the middle. Aldetetrose have two chiral atoms, C2 and
C3. Note that: For sugars, all of the carbon in the
middle has a hydroxyl group—polyhydroxy
structure. So there two-squared or four possible
stereoisomers because you can create a mirror image
for the two. Two of the isomers have the D-
Aldoses containing from three to six carbon atoms, with
configuration. The two D isomers have the same
the numbering of the carbon atom shown. Note that the
configuration at C3 and the two L isomers have the

figure shows only half the possible isomers. For each If OH is projected upward, it is beta.
isomer shown, there is an enantiomer that is not shown,
the L-series.
• Aldopentoses have three chiral carbons.
WHAT HAPPENS IF A SUGAR FORMS A CYCLIC
MOLECULE?
• Sugars (especially 5-6 C) normally exist as cyclic
molecules.
o Aldopentoses and aldohexoses
o Most carbohydrates are found cyclic rather then
linear
• Cyclization is a result of interaction between
functional groups
o C1 (aldehyde) and C5 (alcohol) forms cyclic
hemiacetal (aldohexoses)
o C2 (ketone) and C5 (alcohol) forms cyclic
hemiketal (ketohexoses)
• Carbonyl carbon becomes a new chiral center:
anomeric carbon
You can see the difference between alpha and beta on
• Cyclic sugar can take two different forms (α and β) its carbon one. In alpha, the hydroxyl is located on the
and are called anomers of each other. right. AS for beta, it is located on the left. You would
have a difficulty in determining the L and D, since the 5 th
carbon is absent with hydroxy group. NOTE: We
establish the L and D if the linear form before the
cyclization process is determined first.
NOTE: HAWORTH PROJECTION FORMULAS. When
Free carbon will be attracted more on the alcohol. It is an
you look at the different isomers of a particular carbon of
electrophilic attack then you have a free-floating
a particular monosaccharide, it depends entirely from the
hydrogen which will attach to the oxygen, resulting in an
second carbon until its ultimate carbon. Glucose is
hemiacetal.
different from aloes—an epimer.
D-glucose. The 1st carbon will react on the 5th carbon, then
the cyclization process occurs. The proximity of carbon 5
to carbon 1 will allow the cyclization process, forming Fischer projection formulas of three forms of glucose.
hemiacetal. Note that the α and β forms can be converted to each
Double bond will be lost. The H can be projected other through the open-chain form. The configuration at
downwards or upwards depending on its orientation on carbon 5 determines the D designation.
pace before the cyclization occurs. Since it is a • Fischer projection formulas are useful for describing
hemiacetal, relatively unstable, it could reverse to its stereochemistry of sugars in their linear forms
original form and undergo another cyclization process, • Haworth projection formulas are more useful in
yielding another anomer. describing cyclic structure.
If the OH is projected below—it is an alpha. o Structures are viewed from the edge of the plane.

▪ Imagine you have a Piattos, which
resembles your Glucose, then put it on your
palm. Look at it at a 45-degree angle below
your eye.
o Five-membered rings are called furanose
o Six-membered rings are called pyranose
A comparison of the Fischer, complete Haworth, and

Haworth representations of sugar structures. (a) A abbreviated Haworth representations of α- and β-D-
comparison of the structure of furan with Haworth glucose (glucopyranose) and β-D-ribose
representations of furanoses. (b) A comparison of the (ribofuranose). In the Haworth representation, the α-
structure of pyran with Haworth representations of anomer is represented with the OH group (red)
pyranoses. (c) α-D-Glucopyranose in the Haworth downward, and the β-anomer is represented with the OH
representation (left), in the chair conformation (middle), group(red) upward.
and as a space-filling model (right). We usually use the D-configuration because it is the most
• Glucopyranose exists in the chair confirmation rather abundant form of glucose in the body.
than the Haworth confirmation. This is still cyclic REACTIONS OF MONOSACCHARIDES
though for Hayworth, we use Hayworth just to even WHAT ARE SOME OXIDATION-REDUCTION
out the edges. For chair confirmation in which glucose REACTIONS OF SUGAR?
or mostly six carbon sugars exist in the body. • Redox reaction of sugars plays key roles in
biochemistry
o Oxidation: releases the energy stored in
carbohydrates, takes place in cellular respiration
o Reduction: forms the carbohydrate, takes place
in photosynthesis and in certain species of
bacteria and soils
▪ Forms the glucose and fructose
• Oxidation reaction of sugars are used to identify the
sugar (laboratory practices)
o Aldehydes can be oxidized to give carboxyl group
of acids (-COO-), used to test for aldoses
▪ COH group found in carbon one of your
structure
▪ When aldehyde is oxidized, some oxidizing
agent must be used.

o Aldoses are called reducing sugars ▪ Deoxy sugars means one of the alcohols or
▪ Because they used up the oxidizing agent one of the hydroxyl groups in the structure
▪ Ketoses can also be reducing sugars does not contain oxygen.
because they isomerize to aldoses ▪ A hydrogen atom is substituted by one of the
o In the cyclic form, the compound formed by the hydroxyl groups of the sugar and one of these
oxidation of aldose is called a lactone. oxy sugars is L-fucose or what we call 6-
• Two types of reagents are used to detect the Deoxy-b-L-galactose, a sugar found in the
presence of reducing sugars: silver ammonia carbohydrate portions of the glycoproteins,
complex ion, Ag(NH3)2+ (Tollen’s test) and glucose including the blood group antigens.
oxidase (which is specific only for glucose but not o Another deoxy sugar, 2-Deoxy-b-D-ribose, is
other reducing sugars). found in DNA
Structures of two deoxy sugars. The structures of the

parent sugars are shown for comparison.
Oxidation of a sugar to a lactone. An example of an • When the carbonyl group of a sugar is reduced to a
oxidation reaction of sugars: oxidation of α-D-glucose hydroxyl group, the resulting polyol is known as an
hemiacetal to give a lactone. Deposition of free silver as alditol. C=O C→OH
a silver mirror indicates that the reaction has taken place. o CO is reduced to a hydroxyl group; hydrogen is
• The H from the OH is removed and becomes a added. Hydrogenation is a form of a
ketone. Silver ions is also yielded. Ag2+ will float reduction—the resulting polyol is called an
around the solution until it encounters the surface of albitol.
the tube inside until it forms a silvery mirror complex.
• If you see a silver mirror in an unknown test—
Tollen’s test because it is an indication of the yielding
of silver ions from your agent to form this lactone.
Two well-known alditols are economically important:

xylitol and sorbitol (e.g., artificial sweeteners)
VITAMIN C IS RELATED TO SUGARS
• Also known as ascorbic acid
• Vitamin C is an important lactone. It is related to
sugars because it is for those organisms that
synthesize Vitamin C. They usually derive Vitamin C
A silver mirror produced by an aldehyde. After the from the carbohydrates that they take in.
addition of Tollens reagent to an aldehyde, a silver • Vitamin C is an unsaturated lactone with a five
mirror has been deposited in the inside of this test tube. membered ring structure.
WHAT ARE SOME OXIDATION-REDUCTION
REACTION OF SUGARS?
• There are also reduced sugars (oxidizing sugars) that
are biochemically important
o A deoxy sugar, L-fucose, is found in the
carbohydrate portions of glycoproteins including
There is a characteristic of double bond, a ketone, and
the ABO blood-group antigens
an esterified oxygen which is a characteristic of a
lactone.
• Most animals synthesize Vit. C except for guinea pigs
and primates, including humans.
o Our metabolism does not synthesize Vitamin C
so we have to take them orally.
• Ascorbic acid is taken through diet; deficiency leads
to scurvy The formation of a phosphate ester of glucose. ATP is
o It is a disorder characterized by dysfunctional the phosphate group donor. The enzyme specifies the
collagen, involving weakened collagen (affecting interaction with —CH2OH on carbon 6.
the hair, teeth, skin) This is present during glycolysis.
o Scurvy: collagen (protein) has structural defects, WHAT ARE GLYCOSIDES, AND HOW DO THEY
causing skin lesions and fragile blood vessels. FORM?
Hydroxyproline (amino acid derivative) is • It is possible for a sugar hydroxyl group (ROH)
important for the stability of collagen. bonded to the anomeric carbon to react with another
▪ Hydroxyproline is located in the crosslinks of hydroxyl group (R’-OH) to form a glycosidic linkage
the collagen. So, removing it can cause (R’-O-R), or a compound known as a glycoside.
collagen to weaken. o Glycosidic linkage is not an ether
o Ascorbic acid is essential for the activity of prolyl o The hydrolysis of glycosidic linkages results to
hydroxylase (enzyme), which converts proline to the original alcohol, not a new compound.
hydroxyproline. • The bond formed is called a glycosidic bond.
▪ Adding hydroxyl to create hydroxyproline
then being synthesized in the cells
o Lack of ascorbic acid affects this activity, leading
to the fragile collagen responsible for the
symptoms of scurvy
WHAT ARE SOME IMPORTANT ESTERIFICATION
REACTIONS OF SUGAR?
• Hydroxyl groups of sugars can react with acids and An example of the formation of a glycoside. Methyl alcohol
derivatives of acids to form esters. (CH3OH) and an α-D-glucopyranose react to form the
• Phosphate esters are important because they are the corresponding glycoside.
usual intermediates in the breakdown of We usually omit the H in writing Haworth projection, the
carbohydrates to provide energy. same way with line angle formula. If you react which is a
hemiacetal with a methyl alcohol, you have CH 3OH. The
picture below will show the process.
The hydroxyl group or any hydroxyl

group in the structure can react with acids and derivatives
of acids to form esters. You will get H-O-R, connecting the
sugar with its derivative, phosphate esters meaning the
molecule attached here is PO3-. So, PO3- is the
intermediate in the breakdown of carbohydrates to
provide.
o Formed by the transfer of phosphate from ATP to
give phosphorylated sugar and ADP.
• These reactions are important in carbohydrate
metabolism.

If you hydrolyze or
reintroduce water, CH3OH will reform, and H will bind with
O. That is the intermediate that needs the lowest energy A disaccharide of β-D-glucose Both anomeric carbons
unless you are going to introduce another oxygen (C-1) are involved in the glycosidic linkage.
molecule, creating a carboxylic acid. The formation of In here, you have the same monomeric units, repeating
glycosidic bond is important because these glycosidic monomeric units (alpha will link to alpha).
bonds between monosaccharides are basis for • Chemical nature of oligosaccharides and
polysaccharides and oligosaccharides’ formation. polysaccharides depend on
• Glycosidic bonds formed through the linkage of the O o 1. the monosaccharides that are linked together
atom of one sugar to another are called O-glycosidic ▪ If it is a glucose, galactose, alose, fructose or
bonds ribose.
o N-glycosidic bonds can be found in nucleic acids, o 2. the particular glycosidic bond formed
especially between the nitrogenous bases and • Example: the difference between cellulose and starch
the five-carbon sugar depend on the glycosidic bond formed between
o S-glycosidic bonds are found in thioglycosides, glucose monomers.
where O is replaced by sulfur o Variation in glycosidic linkages can form linear
▪ It comes in fewer quantity compared to N- and branched chain polymers
and O-glycosidic.
• Glycosides derived from furanose (5 C): furanosides
• Glycosides derived from pyranose (6 C): pyranosides
• Glycosidic bonds between monosaccharides are the
basis for the formation of oligosaccharides and
polysaccharides The linear polyglucose chain occurs in amylose. All
• Glycosidic linkage can take various forms; numerous glycosidic bonds are (1 → 4).
combinations are found in nature. Amylose is a type of starch. All glycosidic bonds that form
• The hydroxyl groups (-OH) are numbered so that they in amylose are alpha one for glycosidic bonds.
can be distinguished; the numbering follows the
carbon atoms.
Two different disaccharides of α-D-glucose. These

The branched-chain polymer occurs in amylopectin and
two chemical compounds have different properties
glycogen. Branched polyglucose-chain glycosidic bonds
because one has an α(1→4) linkage and the other has an
are (1 → 6) at branched points, but all glycosidic bonds
α(1→6) linkage.
along the chain are (1 → 4).
You will know if it is a beta if it goes straight or OH is
Every 10th or 4th monomeric residue, you will find a 1 → 6
projected upwards. These linkages are used to determine
glycosidic bond. This will indicate a branching off and will
the reactivity nature and structure of oligosaccharides and
continue towards how many molecules.
polysaccharides. Properties of them will rely on the
existing glycosidic bond between your monomeric units.

• In testing the presence of reducing sugars, the
anomeric carbon is usually involved in the
reaction. It is also frequently involved in the
glycosidic linkage.
• Note that the internal anomeric carbon in an
oligosaccharide or a polysaccharide is not free to
give a positive result for reducing sugar.
• Only if the end residue is a free hemiacetal rather than
a glycoside will there be a positive test for reducing
sugar.
o Even though a particular polysaccharide or The structures of N-acetyl-β-D-glucosamine and N-
oligosaccharide contains a reducing sugar such acetylmuramic acid.
as glucose, if it is found in the chain, the anomeric
carbon where the aldehyde is found is not
available. REASON: It participates in the
formation of the glycosidic chain.
Reducing sugars. A disaccharide with a free hemiacetal

end is a reducing sugar because of the presence of a free Some hexose derivatives important in biology. In
anomeric aldehyde carbonyl or potential aldehyde group. amino sugars, an -NH2 group replaces one of the -OH
Even though, glucose participates in the formation of a groups in the parent hexose. Substitution of -H for -OH
glycosidic bond then there would be no positive result for produces a deoxy sugar; note that the deoxy sugars
the presence of a reducing sugar. shown here occur in nature as the L-isomers. The acidic
WHAT ARE SOME OTHER IMPORTANT DERIVATIVES sugars contain a carboxylate group, which confers a
OF SUGARS? negative charge at neutral pH. D-glucono-δ-lactone
• Amino sugars: carbohydrates that contain amino results from formation of an ester linkage between the C-
group or its derivatives that substitutes for the 1 carboxylate group and the C-5 (also known as the
hydroxyl group of the parent sugar carbon) hydroxyl group of D-gluconate.
o In N-acetyl amino sugars, the amino group itself
carries the acetyl group (CH3-CO-) as a
substituent
• Important examples of amino sugars: N-acetyl-β-D-
glucosamine and N-acetyl-β-muramic acid. These
are components of bacterial cell walls.
o If you try to determine bacteria, you should be
able to choose the correct stain for a particular
bacteria.

SOME IMPORTANT OLIGOSACCHARIDES • Familiarize yourselves with the connections of the 3
• Oligomers (Grk. oligos = few) of sugars are usually main monosaccharides to form the disaccharides.
disaccharides formed by linking two
The structures of several important disaccharides.
monosaccharides units.
Note that the notation —HOH means that the
• Three of the most important examples of
configuration can be either α or β. When a D sugar is
oligosaccharides are disaccharides
drawn in this orientation, if the —OH group is above the
o Sucrose
ring, the configuration is termed β. The configuration is
o Lactose
termed α if the —OH group is below the ring. Also note
o Maltose
that sucrose has no free anomeric carbon atoms.
WHAT MAKES SUCROSE AN IMPORTANT
COMPOUND?
• Sucrose (table sugar) is extracted from sugar cane
and sugar beets.
• Monosaccharide units: α-Dglucose and β-D-fructose.
• C-1 of the glucose is linked to the C-2 of the fructose
in a α,β(1 → 2) glycosidic linkage.
• NOT a reducing sugar, but monomeric substituents
are.
• If you try to test for reducing sugars for sucrose (the

disaccharide in the image above), it will not yield a
positive result because the anomeric groups are not
free for a carboxyl group to yield a positive result.
• Consumption leads to hydrolysis of monomers which
are then used as energy sources.
• SUCROSE leads to the hydrolysis of monomers
which are then used as energy sources. While
GLUCOSE is directly involved in glycolysis whereas
fructose is isomerized to glucose so there needs to be
an energy input before fructose is utilized by the body.
• Humans consume large quantities (which can lead to
health problems); excessive consumption contribute
to health problems which led to a search for other
sweetening agents.
o Fructose: sweeter than sucrose, but changes
texture of food
o Saccharin and cyclamates: causes cancer in lab
animals
o Aspartame: suspected to cause neurological
problems

ARE THERE ANY OTHER DISACCHARIDES • Yeast contains enzymes that hydrolyze starch to
IMPORTANT TO HUMANS? maltose then glucose, which is fermented during
brewing.
THE CASE OF LACTOSE INTOLERANCE
• Sugar intolerance results from the inability to either
digest or metabolize certain sugars, usually due to
missing or defective enzymes.
• Some adults have a deficiency in the enzyme lactase
in the intestinal villi.
• Adults without the enzyme LACTASE, has Lactose
Intolerance
• LACTASE – an enzyme that hydrolyzes lactose into
• Lactose: made up of β-D-galactose and D-glucose its component galactose and glucose in the
linked at β(1→4) INTESTINAL VILLI in the digestive system.
• A reducing sugar because the anomeric carbon of
glucose is free.
• The ANOMERIC CARBON (the carbon on the
rightmost tip of the glucose in the image above) is
free, as opposed to containing an aldehyde. So it is
free to yield a carboxyl group for a positive result. It’s
free to yield a lactone.
• Have two anomeric forms, each must have the β-D-
galactose while the D-glucose can be either α or β.
• Build-up of lactose causes bacterial lactase to act on

it, releasing H, CO2, and organic acids instead of
galactose and glucose.
• Maltose is obtained from the hydrolysis of starch • The bacterial products lead to digestive problems
• Consist of two residues of D-glucose in an α(1→4) such as bloating and diarrhea; the products further
linkage. draw water via osmosis thus aggravating the
• Differs from cellobiose (from cellulose) only in the diarrhea.
glycosidic linkage which is β(1→4)
• Mammals can digest maltose BUT NOT cellobiose.
o The chemical reactivity of your sugar depends
entirely (especially for polysaccharides) on the
type of monomeric units or the type of glycosidic
linkage.
o Ex. the difference between maltose and
cellobiose is just the alpha and beta, yet they are
entirely different in terms of digestibility in
mammals.

o Heteropolysaccharide: polymer consisting of
more than one type of monosaccharide.
• Complete characterization of a polysaccharide
includes
o Specification of which monomers are present
o Sequence of monomers
o Specific type of glycosidic linkage
HOW DO CELLULOSE AND STARCH DIFFER FROM
ONE ANOTHER?
• Cellulose is the major structural components of plant
cell walls.
o Linear homopolysaccharide of βD-glucose, with
all residues linked in β(1→4) glycosidic bonds.
o Individual polysaccharide chains are hydrogen-
bonded together.
• Animals are not capable of hydrolyzing cellulose
• Even if lactase is present, other people cannot
because of the absence of cellulase.
metabolize galactose.
o Cellulase attack the β(1→4) glycosidic bonds
• If an enzyme that catalyzes the reaction pathway for
o However, Certain bacteria have cellulase,
galactose is absent, galactose builds up resulting to
including bacteria found in termite gut and
galactosemia.
ruminants.
o Severe problem in infants because galactose is
▪ Ruminants – animals that have a four-
converted to GALACTITOL, which cannot be
chambered gut/stomach. They have bacteria
excreted. The accumulation draws in water and
in their gut that can degrade cellulose into
causes swelling, edema causes damage
glucose. Ex. cows, goats
o The critical tissue here is the brain, when the
o Again, the disaccharide that is yielded from the
galactose builds up in the brain, it is critical.
metabolism of cellulose is cellobiose
• Dietary therapy for lactose intolerance and
galactosemia are different.
o Lactose intolerant people must avoid taking in
lactose throughout their lives.
▪ Permanent
o Galactosemia-afflicted people must avoid milk
during their childhood. The development of
metabolic pathways for galactose during puberty
alleviates the problem in most individuals.
▪ Mostly only during childhood
STRUCTURES AND FUNCTIONS OF
POLYSACCHARIDES
• So we have oligomers, which are few repeating units
of monosaccharides. Now we have long chains of
monosaccharides.
• When many monosaccharides are linked together,
the result is a polysaccharide.
• Polysaccharides that occur in organisms are usually
composed of a very few types of monosaccharide. The polymeric structure of cellulose. β-Cellobiose is
o Homopolysaccharide: polymer consisting of the repeating disaccharide. The monomer of cellulose is
only one type of monosaccharide. the β-anomer of glucose, which gives rise to long chains
▪ Ex. glucose, glucose, glucose, glucose, that can hydrogen-bond to one another.
glucose, … OR galactose, galactose,
galactose, galactose, …

• Starch are polymers of α-D-glucose. Has different o Debranching enzymes that attack α(1→6) are
forms; can be distinguished from one another by their used by plants and animals.
degrees of chain branching.
o Amylose: linear polymer linked at α(1→4)
glycosidic bonds
o Amylopectin: branched chain polymer with
branches starting at α(1→6) linkages along the
chain of α(1→4)
The structure of starch is based on the α-anomer of

glucose. The monomer of starch is the αanomer of
glucose, which gives rise to a chain that folds into a helical
form. The repeating dimer has α(1→4) linkages
throughout.
Amylose and amylopectin are the two forms of starch. The starch–iodine complex. Amylose occurs as a helix
Note that the linear linkages are α(1→4), but the branches with six residues per turn. In the starch– iodine complex,
in amylopectin are α(1→6). Branches in polysaccharides the iodine molecules are parallel to the long axis of the
can involve any of the hydroxyl groups on the helix. Four turns of the helix are shown here. Six turns of
monosaccharide components. Amylopectin is a highly the helix, containing 36 glycosyl residues, are required to
branched structure, with branches occurring at every 12 produce the characteristic blue color of the complex.
to 30 residues. HOW IS GLYCOGEN RELATED TO STARCH?
• Starch is to plants as glycogen is to animals: both are
storage polymers.
• Glycogen is a branched-chain polymer of α-D-
glucose; similar to the amylopectin part in this regard.
o Glycogen is more highly branched, every 10th
residue is a branch compared to every 25th in
amylopectin
• Starch are storage molecules and therefore should o Average chain length is 13 glucose residue, and
have a mechanism for releasing glucose. 12 layers of branching
o Enzymes can hydrolyze these different forms of o Has glycogenin at its core, an enzyme that acts
starch: α and β-amylase. as a primer to convert glucose into glycogen.
o β-amylase is an exoglycosidase, cleaves from the • Found in animal cells in granules, observed in liver
non-reducing end of the polymer, produces and muscle cells.
maltose.
o α-amylase is an endoglycosidase, cleaves
anywhere along the chain to produce glucose and
maltose.
• Amylose can be completely degraded to glucose and
maltose by the two amylases; amylopectin is not
completely degraded because the branches are not
attacked.
o The branches that contains the α(1→6) glycosidic
bonds cannot be hydrolyzed by the alpha and
beta amylase. So instead of using alpha and beta
amylase, plants and animals actually use
A comparison of the degrees of branching in amylopectin
debranching enzymes.
and glycogen.

• Plays a structural role, has a fair amount of
mechanical strength because of hydrogen bonds.
o Major component of the exoskeletons of
invertebrates; cell walls of algae, fungi, and yeast.
A core protein of glycogenin is surrounded by branches

of glucose units. The entire globular complex may
contain approximately 30 000 glucose units.
• The number of branch points in the glycogen structure
is significant for two reasons:
o 1. a more branched polysaccharide is more
The polymeric structure of chitin. N-Acetylglucosamine
water-soluble; the more glycogen in a solution is,
is the monomer, and a dimer of N-acetylglucosamine is
the faster energy can be released.
the repeating disaccharide.
o 2. When an organism needs energy quickly for
WHAT ROLE DO POLYSACCHARIDES PLAY IN THE
spontaneous reflexes, the enzyme glycogen
STRUCTURE OF CELL WALLS?
phosphorylase has more potential targets if there
are more branches. This allows quicker • Heteropolysaccharides are major components of
mobilization of glucose bacterial cell walls.
WHAT IS CHITIN? o They are also cross-linked by peptides in
prokaryotic cells walls.
o The repeating polysaccharide units are consist of
two residues held at β(1→4) like cellulose.
o Residues are N-acetyl-D-glucosamine and N-
acetylmuramic acid.
• N-Acetylmuramic acid is found only in prokaryotic cell
walls (therefore in ALL BACTERIA); does not occur in
eukaryotic cell walls.
• Chitin: polysaccharide similar to cellulose in both

structure and function.
o Linear homopolysaccharide with all residues
linked in β(1→4) glycosidic bonds
• Differ from cellulose in the monomeric unit.
o cellulose: β-D-glucose
o chitin: N-acetyl-β-D-glucosamine

The structure of the peptidoglycan of the bacterial cell

wall of Staphylococcus aureus. (a) The repeating
disaccharide. (b) The repeating disaccharide with the
tetrapeptide side chain (shown in red). (c) Adding the
pentaglycine cross-links (shown in red). (d) Schematic
diagram of the peptidoglycan. The sugars are the larger
spheres. The red spheres are the amino acid residues of
the tetrapeptide, and the blue spheres are the glycine
residues of the pentapeptide.
• Plant cell walls consist largely of cellulose.
• Pectin, another component of plant cell walls, is
composed mostly of D-galacturonic acid (a derivative
of galactose) residues.
o Extracted from plants; used as a gelling agent in
yogurt fruit preserves, jams and jellies
• Lignin (L. lignum, “wood”) a nonpolysaccharide
component of plant cells walls; a polymer of coniferyl
alcohol, very tough and durable
o Actually not a polysaccharide but it is interesting
to know about this. When you are chopping wood,
it’s hard not because of cellulose but because of
lignin.
DO POLYSACCHARIDES PLAY ANY SPECIFIC
ROLES IN CONNECTIVE TISSUES
• Glycosaminoglycans: polysaccharide based on a
disaccharide in which one of the sugars is
o An amino sugar
o One of the sugars has a negative charge because
of a sulfate or a carboxyl group
• Involved in a wide variety of cellular function and
tissue.
o Heparin: a natural anticoagulant that prevents
blood clots
o Hyaluronic acid: a component of vitreous humor
of the eye and the lubricating fluid of the joints
o Chondroitin sulfates and keratan sulfate:
component of connective tissues
o Glucosamine sulfate and chondroitin sulfate:
OTC drugs that help repair damaged cartilage
Glycosaminoglycans, which are formed from repeating

disaccharide units, often occur as components of the
proteoglycans.
*Each polymer is classified as a homopolysaccharide

(homo-) or heteropolysaccharide (hetero-). †The
abbreviated names for the peptidoglycan, agarose, and
hyaluronate repeating units indicate that the polymer
contains repeats of this disaccharide unit. For example, in
peptidoglycan, the GlcNAc of one disaccharide unit is
(1n4)-linked to the first residue of the next disaccharide
unit.

GLYCOCONJUGATES: PROTEOGLYCANS,
GLYCOPROTEINS, AND GLYCOLIPIDS
WHAT ARE GLYCOCONJUGATES?
• Aside from being energy storage and structural
materials, sugars are also information carriers:
o They serve as destination labels for some
proteins
o They serve as mediator of specific cell-cell
interaction and interaction between cells and
extracellular matrix
• Other roles of specific carbohydratecontaining
molecules include The transmembrane proteoglycans. The four
o cell-cell recognition syndecans in the center are full-time proteoglycans that
o cell adhesion are usually substituted with heparan sulfate chains (dark
o cell migration blue) toward their N termini. Syndecans-1 and -3
o blood clotting sometimes possess additional chondroitin sulfate chains
o immune response, (pink). The melanoma chondroitin sulfate
• When an informational carbohydrate is covalently proteoglycan/NG2 (green) has one chondroitin sulfate
bonded to a protein or lipid, it forms a chain and is also a full-time proteoglycan. The remainder
glycoconjugate. are part-time proteoglycans. The alternately spliced
WHAT ARE THE DIFFERENT GLYCOCONJUGATES? extracellular segment of CD44 may bear one heparan
• Glycoproteins: have one or several sulfate chain. Neuropilin-1 and betaglycan can be
oligosaccharides covalently joined to a protein. substituted with either heparan or chondroitin/ dermatan
o The oligosaccharides have various complexity sulfate (purple)
depending on the molecule. • Proteoglycans: macromolecules of the cell surface
o Outside cell: on the outer face of the plasma of extracellular matrix, a type of glycoprotein
membrane, in the extracellular matrix, and blood o Unique from other glycoproteins because of
o Inside cell: Golgi complexes, secretory granules, glycosaminoglycan.
lysosomes o One or more glycosaminoglycan chains are
• Highly rich in information; forms specific sites for joined covalently to a membrane protein
recognition and high-affinity binding by other proteins. o Glycosaminoglycan moiety of the proteoglycan
dominates the structure; often the main site of
biological activity
▪ Moiety = is the part of a specific structure
o Major components of connective tissues such as
cartilage
Carbohydrates present in the plasma membrane as short,

sometimes branched, chains of sugars attached either to
exterior peripheral proteins (forming glycoproteins) or to
the polar ends of phospholipid molecules in the outer lipid
layer (forming glycolipids).
• Glycolipids: membrane lipids in which the
hydrophilic head groups are oligosaccharide
o Act as specific site for recognition by
carbohydrate-binding proteins.

o Gangliosides are specific type of glycolipid and o N-Acetylgalactosamine is found at the
the carbohydrate moieties are available for signal nonreducing end of the oligosaccharide in the
transduction. type-A blood-group antigen
o In type-B blood, α-D-galactose takes the place of
N-acetylgalactosamine
o In type-O blood, neither of these terminal
residues is present
o In type-AB blood, both kinds of oligosaccharide
are present
• If a blood transfusion is attempted with incompatible
blood type, an antigen-antibody reaction takes place
o The antibodies on the recipient recognizes the
antigen specific for the introduced blood from the
donor.
o The characteristic oligosaccharide residues on
the introduced blood serve as the antigen
• Carbohydrates present in the plasma membrane as • A cross-linking reaction occurs; cells will clump
short, sometimes branched, chains of sugars together
attached either to exterior peripheral proteins • Wrong blood transfusion can result to life-threatening
(forming glycoproteins) or to the polar ends of severe reaction
phospholipid molecules in the outer lipid layer
(forming glycolipids).
CARBOHYDRATES AND THE IMMUNE RESPONSE
REFERENCES
• Campbell, M. K. & Farrell, S. O. (2009). Water: The
Solvent for Biochemical Reactions. In Biochemistry
6th Ed. p 37-64. Thomson Brooks/Cole, Belmont CA.
• Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2008).
Lehninger principles of biochemistry. Macmillan.
• Rodwell, V . W., Bender, D. A., Botham, K. M.,
• Some of the most important examples of Kennelly, P . J., & Weil, P. A. (2018). Harper's
glycoproteins are involved in the immune response illustrated biochemistry. New York (NY): McGraw-Hill
(e.g., antibodies) Education.
o Antibodies bind to immobilize antigens that attack
the organism.
• Carbohydrates also play an important role as
antigenic determinants:
o portions of an foreign molecule that antibodies
recognize and to which they bind.
ABO BLOOD GROUPS
• The four blood groups are distinguished based on the
oligosaccharide portion of the glycoprotein on the
surface of red blood cells.
o In all blood types, L-fucose (6-deoxy-L-
galactose) is found

LECTURE | FRANCIS IAN L. SALAVER, RMT, MD | MIDTERM A.Y. 2021 - 2022
LIPIDS
LIPIDS ARE ORGANIC COMPOUNDS
• Chemical compounds in which one or more atoms of
carbon are covalently link to atoms of other elements,
most commonly hydrogen, oxygen, and nitrogen.
• Examples:
o Carbohydrates
o Proteins
o Lipids
TYPES OF LIPIDS: FATTY ACIDS

• Linear chain of C-H bonds that terminate with
carboxylic acid
• Plasma – only few exist as free (unbound) fatty acids
and most are bound to albumin
o Most of them attach themselves to albumin
because blood plasma is 90% water and lipids
are hydrophobic – they do not interact with water.
For them to be transported in the blood, they must
attach themselves to something that interact well
with the water and that is ALBUMIN.
• Some are found as part of triglycerides and
phospholipids
o Since Fatty Acids are the simplest type of lipids,
• What is the element that is unique to proteins? they can be found as part of the more complex
Nitrogen. So if you want to identify an unknown types of lipids such as phospholipids, cholesteryl
compound in the lab and want to know if it is a protein, ester, and triglycerides
just simply look if it has a Nitrogen. • Good source of energy
LIPID CHEMISTRY
• Also known as fats
• Composed of mostly carbon-hydrogen (C-H) bonds
o Rich source of energy
o An efficient way of storing excess calories
• Play an integral part of the cell membrane of human
cells

LIPIDS
• Fatty acids are released and transported through CLASSIFICATION OF FATTY ACIDS
binding with serum albumin • Based on the length (number of carbons)
o Short chain (4-6 carbons)
o Medium chain (8-12 carbons)
o Long chain (more than 12 carbons)
• Based on number of C=C double bonds,

o Saturated
o Monounsaturated
o Polyunsaturated
• The image below shows us that fatty acids are

metabolized in the body. They can produce Acetyl-
CoA which can enter the Krebs Cycle which yields
NADH and FADH2 can proceed with the Electron
Transport Chain. These processes are all related to
ATP production.
FATTY ACIDS – ESTERIFIED WITH THE GLYCEROL

BACKBONE OF TRIGLYCERIDES AND
PHOSPHOLIPIDS

LIPIDS
UNSATURATED FATTY ACIDS CAN BE CLASSIFIED UNSATURATED FATS CAN BE EITHER CIS FATS OR
AS CIS OR TRANS FORMS TRANS FATS
• Cis forms – if the hydrogen atoms near the double • While cis fats are beneficial and can promote good
bonds are on the same side of the chain cholesterol, trans fats are considered harmful to
cardiovascular health, especially those trans fats
which come from unnatural sources (e.g.,
hydrogenated oils in processed foods).
• Natural, or ruminant, trans fats occur in the meat and
dairy from ruminant animals, such as cattle, sheep,
and goats. They form naturally when bacteria in these
animals’ stomachs digest grass.
• Trans forms – if the hydrogen atoms are on the
opposite sides of the chain
TYPES OF LIPIDS: TRIGLYCERIDES

• Glycerol backbone esterified with 3 fatty acids
• Each of the fatty acid can be potentially be different
thus forming many possible structural forms of
triglycerides
• If the fatty acid is in Cis form, it will exhibit “kinking”

(bending on its own). Its kinking gives it a difficult
time in becoming solid in room temperature which is
why they typically exist in liquid form.
HYDROGENATION
• Triglycerides that contain saturated fatty acids pack

more closely and tend to be solid at room temperature
– animal sources
• Most triglycerides from plant sources such as corn,
sunflower seeds, etc. are rich in polyunsaturated fatty
acids in cis forms – oil
• Is the process to convert a liquid oil (with kinks in its

structure) into a solid form in room temperature. Ex.
margarine.

LIPIDS
ANIMALS VS PLANT FATS TRIGLYCERIDE IS LIKE A BALLOON: LARGE BUT
LIGHT
TYPES OF LIPIDS: PHOSPHOLIPIDS

• Same structure as TAG except that they only have
two esterified fatty acids
• Glycerol backbone with 2 fatty acid and 1 phosphate
group on the 3rd carbon
• Amphipathic (hydrophobic fatty acids and
hydrophilic phosphate head)
• Take a look at the glycerol in the left side. OH is basic.

In the formation of an ester bond, the thing that will
be used up are the hydroxyl groups of the glycerol
backbone and the carboxylic acid groups of the
three fatty acids.
• So, there will be a reaction between the alkaline
hydroxyl group and the acidic carboxylic acid
group, resulting in a neutralization. This is why
triglycerides are termed as NEUTRAL LIPIDS.
• Phosphate head groups can be choline, inositol,
serine, and ethanolamine
o The 4 mentioned phosphate-containing groups
that are found attached to the third carbon of the
glycerol backbone in the phospholipids.
• Most of the time, one of the fatty acids is saturated
while the other one is unsaturated
• Hydrophobic – no polar charges or hydrophilic groups

• Neutral lipid
• Good energy source
• Large lipids but light
• 80% of the fats in the diet
o So expect that after ingesting a meal, majority of
the lipids that will be absorbed from the intestinal
lumen to the blood will be triglycerides.
o They are large in volume but relatively light in
weight. They are big but are light.

LIPIDS
• cholic and chenodeoxycholic acid compose the bile
acid. Bile acid is known to be stored in the
gallbladder. In
• Some cholesterol molecules are converted by
endocrine glands to steroid hormones
• Testes will CORner the OVARY
o Testosterone
o Aldosterone
o Cortisol
o Androgens
TYPES OF LIPIDS: CHOLESTEROL o Estrogens and Progesterones
• Composed of 4 fused hydrophobic rings (A,B,C

and D) and 1 hydrophilic hydroxyl group attached to
A ring
• Amphipathic
• Hydroxyl groups oriented outwards while the fused
rings are buried in the cell membrane
TYPES OF LIPIDS: CHOLESTERYL ESTER
• Same structure as cholesterol except that the
hydroxyl group is replaced by fatty acid
• Cholesterol esterified with fatty acid
o You will no longer find a hydroxyl group, instead,
there will be a fatty acid
• Hydrophobic
o 4 rings = Hydrophobic
• Exclusively synthesized by animals (humans)
o Fatty acid in the end = Highly Hydrophobic
o There are also other sterols that other living
• Good source of energy because of the fatty acid
organisms can produce.
o Ex. ergosterol in fungi
• Readily catabolized by cells thus does not serve as
source of energy
• Converted by cholic and chenodeoxycholic acid
which aids in fat digestion
NAME THAT LIPID

• Out of all the complex types of lipids, there are:
o two highly hydrophobic – triglycerides and
cholesteryl ester
o two amphipathic lipids – phospholipid and
cholesterol
LIPIDS
• Made up of glycerol backbone and 3 fatty acids - • Let’s look at the components of the lipoprotein.
Triglyceride o Triglycerides and cholesteryl ester – are highly
• Made up of glycerol backbone, 2 fatty acids and one hydrophobic so expect that you will find them at
phosphate group - Phospholipid the core and central portion of the lipoprotein to
• Made up of 4 fused rings and is highly hydrophobic – hide themselves from water.
cholesteryl ester o Phospholipids and cholesterols – are
• Made up of 4 fused rings and is amphipathic – amphipathic lipids. So they can display
cholesterol themselves in the surface of the lipoprotein
TAKE NOTE: because they have components that can interact
• Majority of the lipids are hydrophobic with water.
• Plasma is composed of 90% water o Apolipoproteins – are also found in the surface
• How are these lipids transported in the blood? of the lipoprotein.
o They must combine themselves with the o Note: if you want to have a hint in knowing where
hydrophilic substances in the blood to be the components are located, remember their
transported. The hydrophilic substances are the properties. Those that are hydrophobic tend to go
PROTEINS. deeper and be closer at the core. While those that
LIPOPROTEIN are amphipathic can be displayed on the surface
• Lipids bind to Proteins (water soluble) = of the lipoprotein.
LIPOPROTEINS
• TAG (Triacylglycerol/Triglyceride) +
Phospholipids + Cholesterol + CE +
Apolipoprotein = LIPOPROTEIN
• Look at the image above. We will discuss theme one

by one.
o Chylomicron – appears to be big. Why?
Because it contains the largest lipid (triglyceride).
Since chylomicron is the largest of the
lipoproteins, expect that it will also be constituted
by the largest type of lipid, triglycerides. They are
good sources of energy because it is made up of
triglycerides.
o HDL – is the smallest among the lipoproteins.
Expect that it will have the least amount of
triglycerides. BUT why is it that HDL, even though
the smallest, is the densest?
▪ Because HDL is considered to contain the
highest amount of apolipoproteins and
proteins are considered to have HIGH
molecular weight.
▪ Generally in lipoproteins, the more protein
component (apolipoprotein) = the denser the
lipoprotein.

LIPIDS
LIPOPROTEINS o If a portion of a apolipoprotein is hydrophobic, it
• Composed of lipids and proteins (apolipoproteins) is inserted deep down the lipoprotein to hide from
• Main purpose is to transport energy -> the core of the water molecules.
lipoprotein essentially represents the cargo LIPID ABSORPTION
transported by lipoproteins • Triglycerides are acted upon by pancreatic lipase
• Size correlates with its lipid content – higher which cleaves them into fatty acids and
o Large lipoproteins have large core regions – TAG monoglycerides/diglycerides
and Cholesteryl esters • How are lipids absorbed?
o The larger the lipoprotein, the higher the lipid o Fatty acids – are generally small. Has no
content than proteins, the lighter it is. problem passing through by passive diffusion
APOLIPOPROTEIN from the lumen of the intestine across the
• Protein portion of lipoproteins intestinal cells to the blood circulation.
• Found on the surface of the lipoproteins o Triglycerides – since they are large. They are
o Help maintain the integrity of the lipoprotein not easily absorbed by the microvilli of the
o Some bind to host cell receptors intestinal cells. Thus, broken down by
o Some are activators or inhibitors PANCREATIC LIPASE.
• Associates with the lipids because of their
amphipathic helix (protein sequences that fold once
in contact with a polar/non-polar interface)
• Proteins are hydrophilic in nature, but why can they

pack themselves with hydrophobic lipids?
o Associates because of the amphipathic helix
o ALSO, even though proteins are generally
hydrophilic, it can also contain hydrophobic
portions due to its nonpolar amino acids.
• What is the action of lipase?
o Breaks down triglycerides into its component
diglycerides and monoglycerides for easier
absorption of the intestinal cells.
• Look at the picture above. Triglycerides that were
broken down were reassembled back into
triglycerides after it has passed through.

LIPIDS
• Phospholipid is acted upon by the phospholipase

enzyme releasing fatty acid and lysophospholipids
• The short chain fatty acids from the digestion of TAG,
phospholipids and cholesteryl esters are are readily
absorbed and are transported by albumin in the blood
• How do we absorb phospholipids?
• Acted upon by the PHOSPHOLIPASE enzyme. The
enzyme will only remove 1 fatty acid, thus, 1 fatty acid
tail is removed. The remaining portion will be called
LYSOPHOSPHOLIPID (head and single fatty acid tail
only).
o The 2 components (head and tail, & tail only) will
simple be absorbed through passive diffusion by
• Cholesterol is absorbed by the action of the bile the intestinal cells.
• Absorption is via the NPC1-L1 receptor o Note: be specific with the term lipase. If it comes
• Cholesteryl ester acted upon by cholesteryl esterase from the pancreas, term it as pancreatic lipase.
producing cholesterol and fatty acid
• What happens when cholesterol is mixed with bile
acids?
o Cholesterol molecule will be mixed with the bile
acids. And the mixing will enable the cholesterol
molecule to be absorbed via the NPC1L1
receptors displayed on apical cells of the
intestinal cells.
• What is the relation of the NPC1L1 receptor to the
management of people with hypercholesterolemia?
o One of the remedies for people with
hypercholesterolemia is to block the NPC1L1
receptor so that cholesterol will not be absorbed
anymore. Thus leading to the lowering of the • Let’s review this picture.
blood cholesterol level. o The triglyceride is in the intestinal lumen. It is then
• What if the lipid included in the diet is cholesteryl acted upon by the pancreatic lipase breaking it
ester? How would it be absorbed? down into smaller components: monoglyceride
o It will be acted first by the CHOLESTERYL and the fatty acids. They are now readily
ESTRASE enzyme from the pancreas. It will absorbed by the intestinal cells.
break the ester bonds, leading to the liberation of o Once inside the cytoplasm of the intestinal cells,
the fatty acids from the cholesteryl ester and it will they are REASSEMBLED.
now be converted into a cholesterol. o Take note, this triglyceride, along with the
o Cholesterol will be mixed with the bile acids and absorbed cholesterol, phospholipid, and
will be absorbed through the NPC1L1 receptor. cholesteryl ester will be assembled in the form of
o What about the fatty acid? It will just pass through CHYLOMICRONS.
by passive diffusion since it is small. o Note: Chylomicrons are only formed if the FOUR
LIPIDS will be in a complex with apolipoprotein.
LIPIDS
So after absorption, these components are intestinal cells towards the lamina propria and it will
assembled inside the intestinal cells into be absorbed in the green-colored lymphatic
chylomicrons. capillaries.
• Let’s look at the component of the chylomicrons. • Chylomicrons will not go directly in the blood. It first
o Since 80% of the lipids in the diet are triglycerides goes in the lymphatic capillaries. Why?
then it will dominate as a main constituent of the o Because they are LARGE lipoproteins and they
chylomicron. cannot diffuse to the walls of the blood vessels.
o Since triglycerides are large lipids, expect that o Lymphatic capillaries are more permeable than
their resulting chylomicrons are also large. blood capillaries. That’s why chylomicrons will
o Since triglycerides are less in weight (also low enter the lymphatic capillaries.
density) expect that the chylomicrons will be low • How do you think chylomicrons are transported to the
in density. cells of the body so that its triglyceride components
CHYLOMICRONS will become energy sources?
• Transport diet-derived lipids
• Contain large amount of Triglycerides = LARGE but
light!!
• Large size enables it to reflect light and account for
the turbidity of the postprandial plasma.
o Make the serum of the patient turbid
o If you just ate, expect that your serum will be
turbid
o If you centrifuge your blood after eating, expect
that the chylomicrons will be deposited at the top
(because they are light). This accounts for
plasma turbidity at the upper portion.
• Light weight causes it to float to the top of the stored
plasma and form a creamy layer
• Good source of energy (fatty acids in triglycerides)
• Take a look at the picture above.

o The chylomicrons that were absorbed from the
diet in the intestine (pertaining to the red circle)
will be transported via the lymphatic capillaries.
o The lymphatic capillaries will later transport the
chylomicrons to the thoracic duct
o Thoracic duct then later drains to the subclavian
vein
o After the drainage to the subclavian vein, the
chylomicrons are now in our blood circulation
• Take a look at the CM (chylomicron) that is formed.

The CM will be released at the basal part of the

LIPIDS
• Refer to the picture above for a quick recap of the

EXOGENOUS PATHWAY journey towards the clearance of the chylomicron.
• The newly synthesized chylomicrons in the intestine o Once in the blood circulation, they will be
are initially secreted into the lymphatic ducts and transported to the tissues. In the capillaries of
eventually enter the circulation by the way of the these tissues, they will interact with the heparan
thoracic duct. sulfate and other proteoglycans, activating LPL
• Now what will happen at the tissue level, now that the (lipoprotein lipase).
chylomicrons have already reached the blood? o LPL acts on the triglyceride components of the
o These chylomicron lipoproteins will bind to chylomicrons, liberating glycerol and fatty acids.
proteoglycans, particularly heparan sulfate. Thus, converting them into energy.
• In the tissues, chylomicrons bind to heparan sulfate o Since the chylomicron will be losing its
and other proteoglycans present in the capillaries of triglyceride components, it will become smaller. It
different tissues. The binding to proteoglycan is now referred to as chylomicron remnant.
promotes interaction with lipoprotein lipase. o Chylomicron remnants are metabolized or
o Take note of the name “lipoprotein lipase.” This cleared by the liver.
specializes in liberating fatty acids from o This is hours after eating, the plasma of the
triglycerides. patient will be cleared (from the chylomicron
o This is why we should properly differentiate turbidity) Why?
lipases (pancreatic lipase and lipoprotein lipase) ▪ Because of the clearance or the metabolism
from one another because they can come from of the chylomicron components of the blood.
different sources. • The entire process of synthesizing chylomicrons and
• What happens in chylomicron lipoproteins interact metabolizing them is called the EXOGENOUS
with lipoprotein lipase? PATHWAY.
o All the fatty acids in the triglycerides will be • Remember: chylomicrons are derived from the lipids
liberated. in the diet.
o The liberated fatty acids will now be used by the o EXO = from external sources (like in the diet)
tissues as sources of energy. o GENOUS = originating
o Also, the glycerol backbone can be used as • Lipoprotein lipase hydrolyzes TAG on the
energy. chylomicrons generating fatty acids and glycerol.
o The next thing that happens will be the loss of the • The fatty acids and glycerol are then taken up by the
triglyceride component of the chylomicron cells and are used as source of energy.
because the triglycerides are already used up by • Excess fatty acids are then stored in adipose tissues
the cells with the help of the lipoprotein lipase. and are stored there as triglycerides
o Since the chylomicron is losing its triglyceride
component (the lipid that is making it big), it will
turn smaller and become a CHYLOMICRON
REMNANT.

LIPIDS
o It starts with the absorption and digestion of
dietary lipids (“dietary lipids” at the bottom left
corner of the pic)
o They then assemble to form the chylomicrons.
o Chylomicrons gets secreted first into the
lymphatic capillaries which will then get
introduced in the blood circulation after the
thoracic duct drains into the subclavian vein.
o At the level of the tissues, the chylomicrons
(particularly triglyceride) will be hydrolyzed by
LPL (lipoprotein lipase).
• Recap (referring to the pic above): o The chylomicron remnant on the other hand, gets
o The excess fatty acids from the hydrolysis of the metabolized by the liver.
triglyceride from the chylomicron will be stored in VERY LOW DENSITY LIPOPROTEIN
the adipose cells to serve as future sources of • Lipoprotein involved in the endogenous pathway.
energy. o Endogenous pathway – the lipids that will it will
o The fatty acids will be stored in the adipocytes as transport will be coming from the body.
TRIGLYCERIDES. • Transports hepatic-derived (liver-derived) lipids
o These lipids are synthesized by the liver in times
of fasting and starvation.
o If you are eating, you are producing chylomicrons
but if you are not, then the body will be producing
sets of lipids.
o The produced lipids will be transported through
VLDL
• Transfers triglycerides from the liver to the peripheral
tissues
• Contains large amount of Triglycerides but not as
much as Chylomicrons = LARGE but light!!
o It is expected to have large amounts of
• Should the body be subjected to fasting or starvation, triglycerides because its purpose is to be an
the stored triglycerides will be broken energy source.
down/catabolized to release the fatty acids. o It is not as big and light as the chylomicrons.
• The fatty acids will be used by the cells as sources of o Since VLDL is a big molecule, it can also block
energy. out light.
• Good source of energy (fatty acids in triglycerides).
• Like chylomicrons, its can also reflect light and
account for the turbidity in fasting hyperlipidemic
plasma
o BUT will not form the creamy layer like that of
chylomicrons
• Excess dietary intake of carbohydrates, saturated
fatty acids and trans fatty acids enhance the hepatic
synthesis of triglycerides.
• What will trigger the liver to produce high amounts of
VLDL?
o The following are the reasons why:
• Summary of the exogenous pathway or the ▪ Excess intake of carbohydrates
absorption pathway (refer to the pic above) ▪ Excess intake of saturated fatty acids from
animal sources

LIPIDS
▪ Excess intake of trans fatty acids from the provides the testes, the adrenal cortex, and the ovary
hydrogenation of plant oils or the cis forms of the cholesterol for them to produce steroid hormones.
the saturated fatty acids • BUT excess LDL can deposit cholesterol in the walls
of the arteries, which is why it is termed as BAD
CHOLESTEROL. But take note, that it is only
considered “bad” if it is present in high amounts.
• Look at the VLDL in the image above. The VLDL will

be acted upon by the lipoprotein lipase (LPL), which LOW DENSITY LIPOPROTEIN
is similar to what happens to chylomicrons. • Transports cholesterol to the different cells in the
• 50% of the VLDL remnants will be transported to the body
liver for metabolism. That’s only 50%, what will • Taken up by the cells by binding to LDL receptors
happen to the other 50%? present in the cells
• Bad” cholesterol
• The other 50% will start accumulating cholesterol in • Look at the picture above. LDL functions to transport
its structure. It will start picking up cholesterol from the cholesterol towards the different cells of the body.
blood or from the tissues and incorporating these How do you think they are able to transport it to the
cholesterol molecules in its structure. cells?
• What will happen? The VLDL remnant will become • Apo B-100 – the green colored portion. The LDL will
the INTERMEDIATE DENSITY LIPROPROTEIN use it to bind to the LDL receptors present on the cell
(IDL). membrane on the human cells.
• This IDL will still continue accumulating cholesterol • The binding of the Apo B-100 to the LDL to the LDL
until such time it will become the Low Density receptor will cause the LDL to be internalized by the
Lipoprotein (LDL). This is why LDL has HIGH cell.
CHOLESTEROL content because it is derived from • The lysosome will then digest the
VLDL catabolism by accumulating cholesterol. engulfed/internalized cell, releasing the cholesterol
• The main purpose of VLDL – is not to transport from it.
triglycerides. But to transport cholesterol. • Cholesterol will be used as part of the cell membrane
• LDL is good (useful) because it provides the cell or be converted to steroid hormones.
cholesterol to be part of the cell membrane. It • Once bound to the receptors, the LDL are
endocytosed.

LIPIDS
• Transported to the lysosomes for degradation start picking up cholesteryl ester and
• TAG component is acted upon by acid lipase to triglycerides. That’s the time that HDL will be
produce glycerol and fatty acids – source of energy formed.
• Cholesterol – may be used for membrane synthesis, o Apo-A1 can activate an enzyme (LCAT) – is not
synthesis of steroid hormones and stored as just there to assemble the HDL, it particularly
cholesteryl esters by the enzyme acyl- activates LCAT (lecithin cholesterol acyl
CoA:cholesterol acyltransferase (ACAT) transferase).
• What is the main role of HDL?
o Transport cholesterol away from the tissues to
lessen the chance for plaque formation. Thus,
reducing the chance for stroke and acute
myocardial infarction.
o It will also transport the cholesterol to the liver for
the conversion of bile, which is the means of
excreting excess cholesterol.
• Summary
• The LDL in the blood will use the Apo B-100 to bind • There are two types of HDL: discoidal HDL and
to the LDL receptors in the cells. It will cause the spherical HDL.
engulfment of the LDL molecule. • Discoidal – the newly-synthesized HDL
• LDL molecule will be enclosed in a vesicle and fuse
with the lysosome for digestion. The cholesterol will
be consumed.
• Some of them will be stored in the form of cholesteryl
ester to be used in the future.
• The remaining small amounts of triglycerides will be
acted upon by acid lipase for the glycerol and fatty
acids to be used as source of energy.
• This is the discoidal form of HDL. It’s primarily
HIGH DENSITY LIPOPROTEIN
composed of Apo-A1. Main function is to pick up
• Of all the lipoproteins, this is the smallest but the
cholesterol and triglycerides in the tissues to transport
densest. Why?
it to the liver.
o Its highest composition is PROTEIN. And
proteins are known to have high molecular
weight.
• Smallest but the most dense lipoprotein
• Transports cholesterol from the cells to the liver for
• You can now see in the image that the cholesterol is
elimination or formation of bile acids
loaded in the HDL.
• Secreted by the liver and small intestine
• These cholesterols might escape from the structure
• “Good” cholesterol
of the HDL. For the HDL to hold on to these
• Has Apo-A1 (activator of Lecithin Cholesterol
cholesterol molecules they must be transported to its
Acyltransferase)
CORE region.
o The Apo-A1 of HDL is produced by the small
intestine and the liver. After the production of
Apo-A1 by the small intestine and the liver, it will
LIPIDS
• Since cholesterol molecules are AMPHIPATHIC, you • This converts now the free cholesterol to cholesteryl
cannot put them in the core region (because mostly, ester. The previous image is what happens to the
hydrophobic particles are there in the core). cholesterol make it into cholesteryl ester to be stored
• Which is why these cholesterol molecules will be in the core region.
esterified with the fatty acids, leading to the REVERSE CHOLESTEROL TRANSPORT PATHWAY
conversion of the green-colored cholesteryl ester. • Excess cholesterol from non-hepatic tissues is
This is the measure for the cholesterol molecules to transferred to the liver for metabolism and excretion
not escape the bind with the HDL. into the bile.
• The resulting cholesteryl ester can now go to the core • Lipid-poor discoid HDL particles, produced in the liver
of the HDL and be secured. or the intestine, initiate the efflux of cholesterol and
• What converts the cholesteryl ester back to phospholipids from cell membranes via interaction
cholesterol? with the adenosine triphosphate-binding cassette
o LCAT enzyme transporter A1 (ABCA1).
• What is the activator of LCAT? o Imagine if a person is born without the ABCA1.
o Apo-A1 o That person will have no influx of cholesterol and
o This is why it is important for HDL to have Apo- phospholipids in the body. Therefore, the
A1 since it is part of its physiology makeup which discoidal HDL will never get cholesterol and
is to transport cholesterol. phospholipids.
• What will happen with the discoidal form of HDL after o Discoidal HDL will use the enzyme lecithin
having its cholesterols converted to cholesteryl ester cholesterol acyl transferase to convert the
to go inside the core? cholesterol to cholesteryl ester. BUT this cannot
o It transforms into the spherical HDL form happen if there is not ABCA1 receptor.
o People will not be able to excrete cholesterol from
their body through the bile.
• Subsequent action of lecithin-cholesterol acyl
transferase (LCAT) esterifies cholesterol in preβ-HDL
particles and converts them to mature α-HDL
particles.
• Recall, recap of the previously-discussed pathways:
o Exogenous pathway – chylomicron
o Endogenous pathway – VLDL (which can give
rise to LDL)
• The image above is now the mature form of the HDL
o Reverse Cholesterol Transport Pathway – HDL
• Lecithin is a phospholipid and with the action of

lecithin cholesterol acyl transferase (LCAT) enzyme,
the second carbon of lecithin will be esterified to the
cholesterol.

LIPIDS
• For the HDL to be recognized as HDL by the liver, the • HDL will then transport the cholesterol that has been
HDL will bind to the SR-B1 receptor. converted into cholesteryl ester to get recognized by
• SR-B1 receptor – Scavenger Receptor Type B1 the SR-B1.
• But the things is, some of the spherical HDL (mature
HDL) will be acted upon by CETP (cholesteryl ester
transfer protein)
o From the name itself, it will transfer the
cholesteryl ester of HDL into VLDL and LDL.
• This is the mature HDL (pointing to the thing labelled

HDL at the middle of the image) and it is supposed to
transport the cholesteryl ester to the liver by binding
to SR-B1.
• But some of these HDL will be acted upon by the
CETP (cholesteryl ester transfer protein). What
• The pictures above recaps the process of HDL from happens next?
the picking up of the cholesterol, to the conversion, up o The cholesteryl ester in the HDL will now be
to the recognition by the liver. transported to VLDL and LDL.
• The VLDL and LDL might help in the elimination of
cholesterol by binding to the LDL receptors present in
the liver.
• The problem is, not all of these VLDL and LDL will
transport the cholesteryl ester to the liver. Some of
them will go to the circulation and bring the
cholesterol and the cholesteryl ester to the tissues.
• So if a patient has hypercholesterolemia, it is better to
give a drug that will inhibit the cholesteryl ester
transfer protein (CETP) so that HDL will no longer
give the cholesteryl ester to the LDL.
• Mature HDL can deliver cholesterol to the liver either
directly via:
o (1) the scavenger receptor type B1 (SR-B1)
• There is a plot twist in the story.
o (2) indirectly by exchange of cholesteryl esters to
• We have here the discoid HDL (pointing to the portion
apoB-containing particles for triglycerides (TG).
labelled as Pre-B HDL). With the help of the LCAT, it
▪ Cholesteryl esters can be exchanged for
will become the mature HDL.
triglycerides in apoB-rich particles (LDL and
VLDL) by cholesteryl ester transfer protein
(CETP).
LIPIDS
▪ The uptake of apoB-rich particles via hepatic o Since the LCAT enzyme is an important
LDL receptors enables the delivery of component of the HDL. Expect that only the HDL
cholesterol to the liver (approximately 50% will contain the Apolipoprotein A1.
of RCT. • major protein component of HDL particles in plasma
TANGIER DISEASE • Chylomicrons secreted from the intestinal enterocyte
• An inherited disorder characterized by significantly also contain apo A1, but it is quickly transferred to
reduced levels of high-density lipoprotein (HDL) in the HDL in the bloodstream.
blood o Chylomicrons will also originally contain Apo A1
• Mutations to chromosome 9q31 lead to a defective but with the encounter with HDL, it will transfer
ABCA1 transporter. These mutations prevent the the Apo A1 to the HDL because Apo A1 is most
ABCA1 protein from effectively transporting useful in HDL.
cholesterol and phospholipids out of cells for pickup 2. APOLIPOPROTEIN B100 (ApoB100)
by ApoA1 in the bloodstream • Apolipoprotein B100
o Mutations result to the loss of efflux cholesterol • Synthesized in the liver along with the synthesis of
and phospholipids into the blood. Therefore, the VLDL
discoidal HDL will no longer receive cholesterol. o Not incorporated in the chylomicron
o Cholesterol will also not be acted upon by LCAT • Important in the assembly of VLDL and cellular
enzyme and there will be no formation of the uptake of LDL
spherical/mature HDL • Present in VLDL, IDL and LDL
o What will happen? • What is the function of the ApoB100?
▪ There will be an accumulation of deposition o The Apo B100 will be used by the LDL to bind with
of cholesterol in the different parts of the the receptors present on the cell membranes of
body. human cells. This is for LDL to be internalized and
• This disease can predispose someone to early onset metabolized.
of acute myocardial infarction. 3. APOLIPOPROTEIN B48 (ApoB48)
• Apolipoprotein B48
• ApoB48 is identical to the amino-terminal 48% of
ApoB100
• involved in the synthesis, assembly and secretion of
chylomicrons
• Only present in the chylomicrons. This is the
lipoprotein that stabilizes the chylomicron molecule.
4. APOLIPOPROTEIN C-II
• Apolipoprotein C-II (pronounced: Apolipoprotein “C2”)
• Activator of lipoprotein lipase
• So what lipoproteins need lipoprotein lipase as part of
• People with this disease will have yellowish
their metabolism?
depositions in their tonsils. It is composed of
o Chylomicron
cholesterol.
o Which is why chylomicrons need to also be
• They would also have increased risk of developing
loaded with Apo C-II aside from ApoB48
plaque formations in their arteries, resulting to
• What is another lipoprotein would also need
peripheral vascular disease, stroke, and coronary
lipoprotein lipase in its metabolism?
heart disease or acute myocardial infarctions.
• VLDL
APOLIPOPROTEIN
• Since LDL and IDL is also derived from VLDL, expect
• Proteins that bind lipids to form lipoproteins
that they would also contain ApoC-II
1. APOLIPOPROTEIN A1 (ApoA1)
• Present in chylomicrons, VLDL, IDL and LDL
• Apolipoprotein A1
5. APOLIPOPROTEIN E
• Produced by the liver and intestine
• Apolipoprotein E
• Activator of LCAT enzyme
• Facilitates the binding of lipoprotein remnants to the
liver for elimination or metabolism

LIPIDS
• Present in chylomicrons and VLDL o Main composition: Triglycerides
o Why is it present in chylomicrons and VLDL? • VLDL
• This is mainly due to the fact that o Main composition: Triglycerides (second highest
chylomicrons and VLDL are the ones that triglyceride composition by weight among the
have remnants when they are acted upon by lipoproteins)
the lipoprotein lipase since they are large o Main Apolipoprotein: apoB100
molecules. • LDL
• How do you remember the locations of the different o Highest cholesterol content among the
apolipoproteins discussed? lipoproteins because it is formed through the
o Apolipoprotein A1 (A = angel) = in good catabolism of VLDL by cholesterol accumulation.
cholesterol (HDL) o Main Apolipoprotein: apoB100 (because it is
o Apolipoprotein B100 (B = bad 100) = in bad derived from the VLDL which also contains
cholesterol (LDL) apoB100)
o Apolipoprotein B48 (not as bad, only 48% bad) = • HDL
in the chylomicron o Has the lowest triglyceride content among the
o Apolipoprotein E (rEmnants) = function to bind lipoproteins
lipoprotein remnants to the liver o Main composition: protein (which is why it is
considered the densest)
o Main Apolipoprotein: apoA1 (because it is
produced in the liver and the ileum, which is the
origin of the apoA1)
• Look at the role of the ApoE in chylomicron and VLDL

catabolism in the image above.
KEY POINTS OF THE TABLE

• Chylomicrons
o Produced in the ileum (part of the small intestine),
then transported through the lymphatic system to
be drained by the subclavian vein down to the
thoracic duct.
o Main Function: to transport diet-derived lipids
o Main Apolipoprotein: apoB48

SUBJECT NAME FIRST SEMESTER
LABORATORY OR LECTURE (REMOVE IF WALA)| PROF. NAME | MIDTERM A.Y. 2021 - 2022
LESSON TITLE
ADULT REFERENCE RANGE FOR LIPIDS o However, a drop of 20% in HDL is seen in in boys
during puberty (same level as male adults)
• Lower HDL cholesterol coupled with higher LDL
cholesterol increases the risk of heart disease in men.
• Genetic and lifestyle are two factors that increases
risk for heart diseases
• Diet – people who tend to eat more animal fat and
more of grains, fruits and vegetables have lower LDL
• National Cholesterol Education Program (NCEP)
LIPID AND LIPOPROTEIN AND POPULATION developed a list of risk factors for heart diseases.
DISTRIBUTION
Low levels of HDL will put you at risk to develop heart

disease. Metabolic syndrome is a combination of the
other risk factors—a person has a diabetes and
hypertension at the same time.
LIPIDS DISORDERS: DIAGNOSIS AND TREATMENT
• Collectively called dyslipidemia
• Characterized by elevation of plasma cholesterol,
triglycerides (TGs), or both, or a low high-density
The graph shows the differences in the cases of
lipoprotein cholesterol level that contributes to the
coronary diseases between men and women of different
development of atherosclerosis and other related
age groups.
diseases
• Blood levels of cholesterol and triglycerides increases
• Can be caused by genetic abnormalities or
with age
environmental factor or lifestyle imbalances
o Circulating levels of total cholesterol, LDL and
• Others occurs secondary to other underlying
TAG in young children are lower than in adults
disorders
• Women have higher HDL and lower total cholesterol
ANTERIOSCLEROSIS
than men due to the hormone Estrogen
o The differences in the HDL and total cholesterol
disappears after menopause
▪ The risk to develop coronary disease
between men and women would almost be
the same.
o If you are going to compare the total cholesterol
of a man and woman of the same age, expect that
the man would have a lower HDL and a higher
total cholesterol, putting the man at a higher risk
to develop coronary heart disease compare to
women.
• HDL levels for boys and girls are comparable to that
of adult women

LESSON TITLE
• Occurs when arteries grow thick and stiff and restrict This is the gross appearance of fatty streaks.
blood flow to organs and tissues in the body Underneath this is a group of macrophages which have
o Resulting in decreased blood supply to the organ ingested deposited cholesterol and cholesteryl ester
that is being supplied by the artery molecules.
o Imagine, coronary arteries of the heart will
become occluded due to arteriosclerosis,
muscles will no longer receive blood supply and
they will undergo necrosis.
• One of causes of death and disability\
o If not treated, the person could die. If the person
is treated, there would still be some level of
disability that will happen to the person. Reason:
cardiac muscles could no longer regenerate so
the ability of the person to do strenuous or
physical activities will be reduced.
• Mortality rate has decreased through time due to
advancements in diagnosis and treatment and
increasing awareness on CHD
• LDL is believed central role in initiating the formation
of plaque on the walls of arteries.
o LDL functions to transport cholesterol towards the
cells or tissues, so excess LDL results cells to
become saturated with cholesterol. It will be
deposited by LDL in the wall of arteries. The
presence of cholesterol and cholesteryl ester
deposits will trigger the migration of the
monocytes towards the wall of the arteries and
their differentiation into macrophages.
• Stems from the deposition of cholesteryl esters in
• LDL is oxidized and is then engulfed by various cells
the arterial walls
especially macrophages
o The presence of those deposited cholesterol
o This alters the gene expression of the
molecules will trigger the migration of the
macrophages and they start synthesizing
monocytes from the blood towards the artery’s
inflammatory mediators
wall. These monocytes will differentiate, forming
▪ Macrophages will start transcribing those
macrophages. Macrophages will try to engulf the
genes and the mRNA is translated the
cholesterol molecules and cholesteryl molecules
proteins will be produced. Proteins are the
in attempt to clear them from the wall of the
different inflammatory mediators so instead
arteries. The group of these macrophages with
of helping the artery by clearing the
fats in their cytoplasm will now be referred as
cholesterol; macrophages will end up
fatty streaks.
releasing inflammatory mediators, causing
• Deposition results to the formation of fatty streaks
injury to where the macrophages are
o Thin streak of fats within macrophages within the
present.
subendothelial spaces
o Referred to as foam cells

LESSON TITLE
The orange-colored structure is where the monocyte will
anchor itself so that it can go to the wall of artery and
become the macrophage. The elaboration of cytokines
by the foam cell will increase the number of those orange-
colored structure (adhesion proteins). Addition of
proteins will result in more monocytes will go to the wall of
the arteries.
In the middle of the picture, you have the macrophage that

was recruited from blood monocytes, and it will try to
engulf the excess LDL or deposited cholesterol and
cholesteryl ester molecules. However, this macrophage
will transform to become foam cells which will produce
inflammatory mediators, causing injury to the walls of
arteries. If there is injury, there would be inflammation.
If there is inflammation, there would be damage to the
wall of arteries. Platelets will be activated. Then, this
would stick to the arterial walls, resulting in the formation
There would be formation of plaque through time and it
of plaques.
will increase in size, resulting in the occlusion of blood
flow.
Monocyte has differentiated to form a macrophage, taking

up the excess LDL/ The macrophage will become foam
cell which will produce a lot of cytokines.
• Injury signals from the growing fatty streaks or plaque
trigger the expression of adhesion proteins which The blood inside the lumen of the blood vessel is in
allow the migration of more macrophages, platelets laminar flow. The presence of plaque will disrupt the
and lymphocytes laminar flow. The movement will become non-laminar
• Chronic inflammation results from repeated cycles of and the blood will exert pressure to the growing
injury plaque. The non-laminar flow will contribute to the
increasing size of plaque. Some plaques will rupture,
producing small thrombi or clots. This might occlude
smaller blood vessels.
• Narrowing of the blood vessel causes the blood to
circulate in a nonlaminar manner under great
pressure and this aggravates the plaque formation
• Some can burst and cause thrombosis
• Final event=occlusion of blood flow

LESSON TITLE
This is a blood vessel with a growing atherosclerotic

plaque. • Peripheral vascular disease – if the plaque develops
in the arteries of the arms or legs
• Coronary Artery Disease – heart
o Acute myocardial infarction if there is a total
occlusion of the blood flow.
• Cerebrovascular disease – brain
o Stroke
• Lipid deposits are most of the time seen in cases of
High serum LDL concentration or decreased HDL
This is the blood vessel under the microscope. There is a
concentration
narrowing of its lumen because of the presence of the
growing plaque • Goal: To Lower LDL concentration
• For every 1% decreased in LDL concentration,
there is 2% reduction in the risk of developing
arteriosclerosis
• For patients with history of previous heart disease,
lowering the LDL below 100mg/dL(2.6mmol/L) is
effective in the stabilization and even in regression of
the plaque.
o Plaque will no longer grow in size preventing the
A – plaque developing on the wall of this blood vessel or
occlusion of the blood vessel. The problem with
arteries.
regression of plaque, some of them might
B and C – This plaque just recently ruptured because it’s
rupture.
actively bleeding so this plaque has thrown thrombi away.
• LDL should be lowered in patients:
o Whose diets are rich in fats
o Who lack daily exercise
o Who are smoking and drinking alcohols
o Have diabetes, hypertension and kidney/liver
diseases
o Are obese
• HDL can be increased by drugs such as
o CETP inhibitors
o Niacin-containing drugs –decreased hepatocyte
production of CETP
o Fibrates –increases hepatic production of apoA-1

LESSON TITLE
▪ If there is high amount of HDL, there would
be higher amounts of cholesterol that will be
delivered to the liver for excretion.
o Statins –HMG-CoA reductase enzyme inhibitor
▪ HMG-CoA reductase enzyme is used by the
cells to produce their own cholesterol.
▪ A cell in the body has two possible sources of
cholesterol. It can get the cholesterol from
LDL in the blood but it can make its own
cholesterol. If this enzyme will be inhibited,
the cell would be forced to get the cholesterol
from LDL lipoproteins in the blood; thereby
with the action or statins, LDL will go down.
In Letter A, bile acid is produced in the liver and is

temporarily stored in the gallbladder in the presence of
fats in the diet. The gallbladder will contract, releasing the
bile. Bile will mix with the cholesterol in the diet and the
bile in the cholesterol will be absorbed towards the blood.
• Total cholesterol can be lowered by:
o Cholestyramine – bile acid sequestrants
▪ This will form a complex with a bile acid so
the bile acid cannot form or interact with
cholesterol. So, bile acid are not absorbed We have here a cell that can produce its own cholesterol
in the blood, reducing the blood cholesterol by metabolizing HMG-CoA with the help of the enzyme
level. HMG-CoA reductase. This HMG-CoA will be converted to
o Ezetimibe –inhibiting the NPC1-L1 transporter Mevalonic acid, which will be utilized by the cell or
which is responsible for the absorption of converted by the cell to cholesterol. Statins will inhibit the
cholesterol HMG-CoA reductase enzyme so the cell could not
produce anymore its own cholesterol. The cell will be
forced to express LDL receptors on its cell membrane,
engulfing or getting the LDL molecules from the blood
lowering the cholesterol level.
• HDL can be increased by drugs such as
o CETP inhibitors – Cholesteryl Ester Transfer
Protein will cause the HDL to transfer its
cholesteryl ester to LDL. Good things if the LDL
will deliver the cholesteryl ester to the liver but the
problem is the LDL will transport the
transferred cholesteryl ester to the tissues,
increasing the change for arteriosclerosis. To
allow the HDL to bind to the liver through SRB-1
Bile acid will interact with cholesterol and the cholesterol receptor and not to transfer its cholesteryl ester to
will be absorbed by the intestinal cells via NPC1-L1 LDL, you have to inhibit the cholesteryl ester
receptor. If cholesterol is not absorbed, resulting in the transfer protein.
level of blood cholesterol to drop. o Niacin-containing drugs – decreased hepatocytes
o Niacin production of CETP
▪ Increase blood HDL ▪ The problem with Niacin, you have to monitor
the liver enzymes of the patient because it
can be hepatotoxic. If there is no CETP , the

LESSON TITLE
HDL will not transfer its cholesteryl ester to ▪ Statins will not work on individuals having
LDL. homozygous FH due to the missing genes for
▪ Can cause flushing the expression of these receptors.
▪ Hepatotoxic o Medicines stimulate synthesis of additional LDL
o Fibrates – increases hepatic production of apoA- receptors which will then cause the removal of the
1 by the liver LDL in the blood
▪ If there is increase in the production of apoA- • Homozygous
1, then you have higher number of discoid o Managed with LDL pheresis
HDL resulting in higher number of spherical ▪ The same with hemodialysis meaning blood
or mature HDL. of the patient is made to go through a
FAMILIAL HYPERCHOLESTEROLEMIA machine in dialysis, removing all the waste
• Inherited, increased cholesterol level in the blood products. In LDL pheresis, the blood of the
particularly LDL hypercholesterolemia patient will go inside the machine, removing
• Autosomal dominant all LDL molecules.
o You only need one abnormal gene from one of HYPERTRIGLYCERIDEMIA
your parents to manifest this condition. • Increased triglycerides in the blood
• Increased risk to heart diseases • Can be caused by genetic abnormality – familial
• caused by mutation in the gene for the LDL hypertriglyceridemia mutation in the LPL enzyme
cholesterol receptor gene or deficiency of apo CII)
o LDL is important in the uptake of LDL from the o Lipoprotein lipase is involved in the metabolism of
blood by the blood by the cells in the body. If the chylomicrons and VLDL. If this enzyme will go
cells in the body cannot express LDL receptors, missing, then chylomicrons and VLDL will no
the LDL in the blood will not be taken up by the longer be metabolized causing their build up in
cells. It will result in atherosclerotic plaque the blood.
formation. o Other cause of abnormality is the mutation for the
• Homozygous – person has two abnormal genes gene that encodes for the apolipoprotein CII,
inherited from both parents causing hypertriglyceridemia. Apolipoprotein CII
o 1:1 million in population is involved in the activation of the lipoprotein
o Total cholesterol 800-1000mg/dL (Normal: 20- lipase.
26mg/dL) • Can be caused by diabetes, renal failure
o First heart attack in their teenage years o Diabetes – glucose molecules are not properly
• Heterozygous FH patients – inherit on abnormal gene utilized by the cells in the body as source of
from one of the parents (there is only reduction in the energy. So, the body will look for other potential
expression of LDL receptors) sources of energy. Fats stored in the adipose
o 1:500 in the population cells or triglycerides will be broken down to form
o Tota cholesterol 300-600 mg/dL glycerol and fatty acids, and they will be secreted
o Become symptomatic in their 20s to 50s; some of in the blood. The blood will transport the fatty acid
them might also die because of familiar and glycerol to the liver, assembling the glycerol
hypercholesterolemia and fatty acids into triglycerides and the liver will
pack these into VLDL molecules.
o Renal failure – depresses the removal of the large
molecular weight constituents, such as
triglycerides from the blood.
o Treated with statins or HMG-CoA reductase

enzyme inhibitors

LESSON TITLE
• The exact mechanism is still unknown.
• Diagnosis of the disorder in a particular patient
requires a family history of premature coronary artery
disease
• The pathophysiological mechanism underlying FCH
is believed to be hepatic overproduction of apoB-
100 containing lipoprotein particles (ie, VLDL and
LDL), resulting in increased plasma total cholesterol,
triglycerides, and apoB levels.
• Can be caused by hormonal imbalance (epinephrine. LIPOPROTEIN(a) ELEVATION
norepinephrine, growth hormone, ACTH, glucagon)
o The written hormones can activate a particular
enzyme that is hormone-sensitive lipase. This
enzyme is found in adipose cells. In the presence
of this lipase, it will break down the triglycerides
stored in the adipose cells. So, there would be a
subsequent release of glycerol and fatty acids in
the blood similar with diabetes mellitus.
Triglycerides will be transported in the blood, Shown on the left side of the screen is one LDL
loaded in the VLDL, resulting in increase hepatic molecule, and on the right side of the screen is
production or synthesis of VLDL. Lipoprotein (a). It is an LDL but there is an additional
• Elevated triglyceride level is seen in coronary heart presence of apolipoprotein (a) or Lp (a) attached to LDL
disease will convert LDL into lipoprotein (a).
o A lot of studies have not yet shown a clear • Lp(a) was first identified and classified as a “low
relationship between hypertriglyceridemia and density lipoprotein variant” over 40 years ago.
coronary heart diseases. Some of the patients • Lp(a) showed it to consist of a LDL covalently bound
who are having coronary heart diseases also to a unique protein called apo(a) encoded by Lpa
have high levels of triglycerides in their blood. gene
• Complication
o Acute or recurrent pancreatitis – enzymes that
are supposed to be released by the pancreas into
the small intestine, particularly in the duodenum,
to digest nutrients in the food will digest the
pancreas itself. The activation of enzymes is
brought by hypertriglyceridemia.
• Treatment The apolipoprotein portion of the lipoprotein (a) is similar
o Dietary modifications in terms of structure and appearance to plasminogen.
o Fibrates – increase HDL levels • Apo(a) is a homologue of plasminogen
o Plasminogen is a protein in the blood that is
converted to plasmin that is capable of breaking
down clots, especially if the tissue has already
repaired itself. Plasmin will remove the clot.
COMBINED HYPERLIPOPROTEINEMIA
• abnormal pattern of human serum lipoproteins in
which levels of low-density lipoproteins (LDL) and
very low-density lipoproteins (VLDL) are elevated.
LESSON TITLE
Since its apolipoprotein (A) has similar homology or o Non-specific reaction
appearance to plasminogen, this lipoprotein (a) will ▪ Solution: do hexane extraction – Hexane will
compete with plasminogen to the building sites in the remove the cholesterol so it would be
blood vessels. So, plasminogen will not be converted to separated from other chemicals in the
plasmin. Therefore, clots will not be broken down. If clot sample.
grew in the atherosclerotic plaque, it can grow bigger.
• Lp(a) has proven to be pathogenic in nature and
involved in the development of coronary heart
disease (CHD)
o Lp(a) has been shown to competitively inhibit the
binding of plasminogen to its receptor on
endothelial cells as well as to its binding sites on
fibrinogen and fibrin.
o Once oxidized, it is engulfed readily by • Enzymatic reaction
macrophages thus acceleration of formation of o Clinical laboratory
fatty streaks o Enzymes are specific
▪ If the type of cholesterol deposited in the o No need to do extraction of cholesterol
walls of the artery is Lp(a), so the engulfment o Reagents are not as dangerous as the acidic
would be faster. Thus, formation of fatty reagents used in traditional methods
streak would be faster.
HYPOALPHAPROTEINEMIA
• There is a decrease level of lipoprotein that has an
alpha apolipoprotein. The absence of ABC A1 will not
allow the formation of spherical or mature HDL from
discoidal HDL.
• Decrease in the circulating HDL
• <40mg/dL
• Increased risks for Coronary heart disease
• A good example is Tangier’s disease It starts with cholesteryl ester will be acted upon by
o Characterized by the absence of ABC A1 cholesterol esterase, breaking the ester bonds linking
receptor that allows cholesterol and the fatty acid to the cholesterol molecules in cholesterol
phospholipids to leak out of the cell so they can esters. After the action of the enzyme, cholesterol will be
become part of the discoid HDL. Treat the patient liberated (FC + fatty acids). The free cholesterol
with Niacin. liberated from cholesterol esters will proceed to the
• Treated with niacin (risk for flushing and liver second reaction (catalyzed by cholesterol oxidase) along
damage) with free cholesterol originally present in the patient’s
LIPID AND LIPOPROTEIN ANALYSIS blood (product: cholest-4-ene-3-one + hydrogen
CHOLESTEROL peroxide). Hydrogen peroxide is a known oxidizing
agent. The 4-Aminoantipyrine, an incorporated dye, is
originally colorless. In the presence of hydrogen
peroxide, it will be converted into a quinone pigment (red
in color). The higher number of free cholesterols in the
patient’s sample, the higher amount of hydrogen
peroxide will be generated by the second reaction. The
higher amount of hydrogen peroxide, the more dye will
be oxidized in token pigments by the enzyme
peroxidase—darker final color will appear in the solution.
• Lieberman–Burchard test
o Cholesterol is treated with sulfuric acid, acetic
anhydride, and acetic acid elicits a green-blue
color
LESSON TITLE
lowest concentration of cholesterol because only few of
the dyes were oxidized by hydrogen peroxide. Cuvette 5
has the highest concentration. We cannot give the
specific number to the patients.
This is the cholesterol reagent used in the enzymatic

determination of total cholesterol. The reagent should
contain cholesterol or cholesteryl esterase. So, all the
cholesterol esters will be converted into free fatty acids
and cholesterol in the patient’s serum. It also contains
Put the cuvette in a spectrophotometer.
cholesterol oxidase that will catalyze the second
reaction. Furthermore, it should contain the dye that will
turn red or produce colors in the presence of the
generated hydrogen peroxide. Transfer the reagent in a
test tube or cuvette.
The cuvette with the colored product will be hit by a light

Pipette specific amount of patient’s serum to measure
coming from a light source. Solutions with color can
the amount of cholesterol molecules present in the
absorb specific types of light. You just have to adjust the
sample.
spectrophotometer depending on what sample is being
processed. The darker the color of the final product, the
higher the amount of light absorbed. Light color absorbs
small amount of light. Spectrophotometer will give the
numerical value.
ENZYMATIC REACTION
• Intensity of color is proportional to the concentration
Dispense the patient’s serum in the cholesterol reagent. of cholesterol
So, the cholesteryl esters will be acted upon by • Interference from reducing agents such as bilirubin
cholesterol esterase liberating free cholesterol which will and Vitamin C
proceed to the second reaction, catalyzed by cholesterol • The problem with this enzymatic reaction for
oxidase. The product is hydrogen peroxide which will determination of total cholesterol is that the last
oxidize the dye present in the reagent to produce chemical reaction is an oxidation reaction, the
colored compound. presence of reducing agents such as bilirubin and
Vitamin C can greatly affect the result because they
will act on the oxidized dye converting diet to the
colorless form. It will not show the exact value of
cholesterol so the cholesterol level will be falsely low.
The left side has the lightest color while the right side
has the darkest intensity of color. Cuvette 1 has the

LESSON TITLE
will only reflect the levels of free endogenous glycerol. In
both procedures, we need to prepare glycerol blank that
will give you an idea how much free glycerol molecules
are in the patient’s serum.
SAMPLE TEST: REAGENT USED CONTAINS LIPASE
ENZYME
• Enzymatic assay
• In most specimens, the endogenous free glycerol
contributes 10-20mg/dL overestimation of
triglycerides
o Recall that the second reaction in the enzymatic
Triglycerides in the patient’s serum will be acted upon by
assay for triglyceride would involve the use of
the lipase so there would be generation of glycerol
glycerol molecules that comes from the first
molecules which will proceed to the second reaction. The
reaction. This free endogenous glycerol will
free endogenous glycerol molecules will become part of
become part of the second reaction. This will be
the second reaction. But, the thing is, you already have
a problem for patients with diabetes and liver
an idea how much these three endogenous glycerol
disorder where there is no utilization of glucose
molecules are in the serum since it was measured already
molecules. So, the body will look for other source
in the glycerol blank. You will just simply deduct the
of energy, breaking down triglycerides stored in
amount of the free glycerol measured in the glycerol
the adipose cells. Then the released glycerol will
blank. Then you will arrive to the amount of glycerol
proceed to the second reaction of the enzymatic
molecules derived from the triglycerides of the patient that
assay for blood triglyceride level—falsely
were broken down by the lipase in the first chemical
elevated result.
reaction.
• About 20% of specimens will have high glycerol
DOUBLE CUVET BLANK TECHNIQUE
content due to diabetes and liver disorder
o Perform “double-cuvet” blank or “single cuvet”
blank
o Glycerol blank – serum is tested without the
addition of lipase
o Sample – serum is tested with the addition of
lipase
GLYCEROL BLANK: REAGENT HAS NO LIPASE
The glycerol blank and the sample test will be measured

at the same time. In this setup, the light coming from the
light source will be first directed towards the glycerol blank
and will be the spectrometer’s reading will reflect the
endogenous free glycerol levels and then the light will be
directed towards the sample test. The reagent will contain
You don’t have the first reaction which is catalyzed by the lipase enzyme. In this test tube or cuvette, it is not only
lipase. In performing double cuvette or single cuvette the endogenous free glycerol will be measured but also
blank technique, we will use a reagent that doesn’t have the glycerol molecules produced from the breakdown of
a lipase so the triglycerides in patient’s blood or serum of triglycerides of the patient by the enzyme lipase. Deduct
the patient will not be broken down into glycerol of fatty the total glycerol measured in the sample test and the
acid. The free endogenous glycerol molecules present amount of the free glycerol measured in the glycerol
in the sample will only react to the reagent. Whatever blank.
result will be seen in the reading, the spectrophotometer
LESSON TITLE
SINGLE CUVETTE BLANK TECHNIQUE
The glycerol blanks and sample tests are not read at the
same time. It is the operator’s choice what to measure
first. Load either the glycerol blank or sample test the wait After centrifugation, HDL will settle at the bottom of the
for the absorbance reading. test tube because it has the highest density among all the
LIPOPROTEIN METHODS lipoproteins and chylomicrons will float on the superficial
layer.
PROTEINS CAN CHANGE ITS CHARGES BASED ON
THE pH
Chylomicrons, VLDL, LDL, and HDL have significant

differences in terms of their density. One of the Take a look at the amino acid on the left side of the
procedures that can be employed in the laboratory to screen. The amino acid was placed in an acidic medium.
separate these lipoproteins from each other is Through their amino group will accept hydrogen ions,
Ultracentrifugation. Sample will be spin at an becoming positively charged. Since amino acids are
exceptionally high speed. HDL has the highest density, building blocks of proteins, we can conclude that proteins
then it will settle at the bottom of the test tube. will become positively changed at an acidic pH. On the
Chylomicron will settle superficially since it is the lightest. right side of the screen, amino acid is placed in an alkaline
ULTRACENTRIFUGATION medium. The carboxylic acid group of that amino acid will
• Ultracentrifugation is a specialized technique used to release hydrogen and hydrogen will counteract the
spin samples at exceptionally high speeds hydroxyl group present in the alkaline medium—
• Uses density adjustments or rate of floatation to becoming negatively-charged.
fractionate lipoprotein classes ELECTROPHORESIS
• Cathode – negative
• Anode – positive
• It is used to separate proteins in the laboratory,
o We will place the proteins on the sample well
since we want them to migrate towards the anode
(positively-charged). We should make these
proteins bear negative charges by exposing the
LESSON TITLE
proteins to an alkaline solution because proteins higher the number of negative charges, the more that
would bear negative charges at alkaline pH. this protein will be attracted to the anode.
Since the proteins are negatively charged, it will • Proteins are separated based on their sizes and
be attracted towards the positively charged number of negative charges. Even if these proteins
electrode which is the anode in the were already separated in the electrophoresis gel,
electrophoresis proteins. there is no way for us to see them because they are
soluble substances.
Apply stain on the electrophoresis gel to make proteins

visible to the naked eye.
After staining: 1 – smallest among them because it was

able to migrate closest to the anode and probably has the
highest number of negative charges. Protein 1 is the
darkest, meaning it has the highest concentration. Protein
4 – heaviest or the largest, has the lowest number of
The smaller the protein is, the faster it will migrate and the
negative charges and least concentration.
closer that the protein will be able to reach the anode.
Image shows how results of electrophoresis are reported.

This is the normal serum electrophoresis result, meaning
albumin is the smallest among them. It migrates closest
and has the highest concentration. Gamma are slow
migrating proteins and these proteins are most of the time
antibodies. Electrophoresis is used to analyze
lipoproteins because they have proteins in their structure.
• Another factor that we need to consider is the amount
HDL will migrate at the alpha region since it contains
of negative charges that the protein would bear. The

LESSON TITLE
apolipoproteins a1. VLDL and LDL will migrate towards We will add poly anions and divalent cations into the
the beta region because both of them contains patient’s serum, precipitating VLDL, LDL, and
apolipoprotein b100. chylomicrons. If the patient did not fast. If the patient
undergoes fasting, then LDL and VLDL precipitated
because chylomicrons are formed from diet derived lipids.
After addition, centrifuge the sample so the sediments
would contain precipitated VLDL and LDL. The
supernatant will contain HDL. Pipette out the supernatant
and test the cholesterol. Through this test, we can
determine the levels of HDL of the patient because we
were able to separate or precipitate out VLDL and LDL.
IMMUNOCHEMICAL METHODS
• Make use of specific antibodies to the apolipoproteins
Thinnest arrow indicates protein 1 representation on the
o I want to separate HDL from other lipoproteins.
curve.
So, I will use antibodies to Apolipoproteins a1.
They will all bind to HDL separating the HDL from
other apolipoproteins for they do not contain
apolipoproteins a1 similar to HDL.
• Antibodies are immobilized in a column matrix or beds
and will capture specific lipoprotein
This is the normal result for lipoprotein electrophoresis.

HDL has the highest protein concentration. Chylomicron
is primarily triglyceride. So, it doesn’t have much protein
to bear negative charges and be attracted towards the
anode.
CHEMICAL PRECIPITATION
• Polyanions such as heparan and dextran sulfate
together with divalent cations such as Magnesium
and Manganese
There are red-colored antibodies. These antibodies are
• Apo-B is rich in positively charged amino acids will
designed to bind to HDL. As you will add the sample of
form complexes with polyanions and will aggregate
the patient into the setup the HDL molecules blue
and become insoluble in the addition of cations
diamond-shaped structures, it will be captured by
o Thus VLDL and LDL are precipitated leaving HDL
immobilized antibodies. The red boxes will remain be
in the solution
suspended in the patient’s sample. To separate the HDL
• Process: we have the patient’s serum, the poly anions
from other lipoproteins, it will be aspirated back the serum
and the divalent cations. The poly anions will be
to separate HDL from other lipoproteins.
attracted to the apolipoprotein b. divalent cations will
HIGH DENSITY LIPOPROTEIN METHODS
precipitate them. In the patient’s serum of the patient,
• Majority of the samples processed for lipid profile are
all of the apolipoprotein b containing lipoproteins will
fasting samples
be precipitated and be left a soluble HDL.
• Chemical precipitation
• Precipitation reagent is added to the serum which will
aggregate and precipitate non-HDL lipoproteins
• Centrifuged at approximately 1500 gravity for 10-
30mins (10,00015,000 g for 3 minutes)
• Supernatant is then separated and HDL is quantified
as cholesterol

LESSON TITLE
o We can use Libermann burchard assay and
enzymatic reaction. We will not perform the
chemical precipitation in total cholesterol
because we want to get the total cholesterol after
determining total cholesterol. After getting the
total cholesterol, we will use a separate serum or
sample of the patient and we will do the chemical
precipitation, separating or precipitating VLDL
and LDL. So, HDL will be processed in the serum. We will have one test tube for total cholesterol, and we
• Chemical precipitants: will have another test tube or set up for HDL determination
o Heparin plus manganese (manganese can inhibit wherein we will perform chemical precipitation and the
enzymes) third setup for triglyceride determination—whatever the
o Phosphotungstic acid plus magnesium (high result will be divided by 5 (VLDL concentration). For LDL,
variability among results) subtract from total cholesterol the VLDL and HDL.
o Dextran sulfate plus magnesium
• Problem: Elevated triglyceride levels can cause
interference
o If VLDL and chylomicrons are present in large
amounts, the low density of aggregated
lipoproteins may prevent them sedimenting and
may even cause floating during centrifugation
▪ If you are going to add the precipitation
agents, they will precipitate out VLDL and
chylomicrons—they have low density and will
float. There is no separation of supernatant
NOTE: triglyceride can be divided by 5 if the unit used is
used to test HDL.
milligrams per deciliter. This formula is not applicable if
o The incomplete sedimentation (seen as turbid,
the triglyceride level is beyond 400. If there would
cloudy or particulate matter) may cause falsely
Hypertriglyceridemia, precipitation of sediments is not
high HDL values
easily separated from the supernatant. If you can perform
o Troubleshooting: ultrafiltration
the ultrafiltration, then this formula can still be applied.
MAGNETIC METHOD FOR AUTOMATED MACHINES
• Similar to Chemical precipitation except that the
precipitation reagent is complexed with magnetic
particle
• The sediments formed do not require centrifugation
• Allows the supernatant to be analyzed without the Concentration of LDL in millimoles per liter
need to remove it from the sedimented complex
Patient is instructed to undergo at least 10 hours of

fasting. Add the cholesterol reagent for enzymatic
reaction to the serum of the patient, and all the cholesterol
from LDL, VLDL, and HDL. In determination of HDL, we
will do the chemical precipitation. Precipitate out VLDL
and LDL leaving behind HDL in the supernatant using
cholesterol reagent so we will be able to measure
cholesterol molecules in HDL. We can measure the
triglyceride level of the patient and divide it by five. The
result will indicate VLDL concentration of the patient.

LECTURE | CHRISTIAN JOHN S. CAPIRIG, MD | MIDTERM A.Y. 2021 - 2022
NUCLEIC ACIDS
REFERENCES
• Lehninger Principles of Biochemistry STRUCTURAL CHEMISTRY OF NUCLEIC ACIDS
• Harper’s Illustrated Biochemistry NUCLEOTIDES, NUCLEOSIDES, AND
• Biochemistry by Campbell NUCLEOBASES
INTRODUCTION
ORGANISM
• Store and preserve information
• Pass information to future generations
• Express information during life’s processes
GENETIC INFORMATION
• Coded along a polymeric molecule (DNA)
o the chemical basis of heredity
• Organized into genes
o units of DNA that encodes a protein or DNA
CENTRAL DOGMA OF MOLECULAR BIOLOGY
• Transcription is carried out by RNA polymerase

• Building blocks of nucleic acids
• Translation is performed on ribosomes
• Nucleotides - have three characteristic components
• Replication is carried out by DNA polymerase
1. A nitrogenous (nitrogen-containing) base (also
• Reverse transcriptase copies RNA into DNA (like HIV
called a nucleobase)
and Hepatitis B virus)
2. a pentose
GENE EXPRESSION & DNA REPLICATION
3. One or more phosphates
“The genetic information stored in the nucleotide
• Nucleoside - Nitrogenous base (nucleobase) +
sequence of DNA serves two purposes.”
pentose only
• Source of information for synthesis of protein
• Nucleobase - nitrogen-containing aromatic
molecules of the cell
compounds that make up the coding portion of nucleic
o GENE EXPRESSION
acids
• Provides information inherited by daughter cells or
• From Biggest to Smallest (-tide, -side, -base)
offsprings
o Nucleotides (nucleobase + pentose + 1 or more
o DNA REPLICATION
phosphates),
GENE EXPRESSION VS DNA REPLICATION
o Nucleoside (nucleobase + pentose),
GENE EXPRESSION DNA REPLICATION o Nucleobase (nitrogen-containing portion only)
Duplicates the NUCLEOTIDE AND NUCLEIC ACID NOMENCLATURE
Produces all the proteins
chromosomes before
required by an organism
cell division
Transcription of DNA: RNA
DNA copy of the entire
copy of a small section of
chromosome
a chromosome
Average size of a human Average size of a
gene: 10 – 10 nucleotide human gene: 108
4 5
pairs nucleotide pairs

Translation of RNA:
*intentionally blank
protein synthesis
Occurs throughout
Occurs during S phase
interphase
Transcription in nucleus;
Replication in nucleus
translation in cytoplasm
NUCLEIC ACIDS
NUCLEOBASES PYRIMIDINE
• Is a heterocyclic
(consists of more
than 1 type of atom)
aromatic organic
compound similar to
benzene and
pyridine consisting
of two nitrogen
atoms as position
1&3 of the six-
member ring.
• Pyrimidine – all pyrimidines have an oxygen double

bonded to their 2nd carbon.
o Cytosine – has an amino group attached to a
carbon
o Uracil – almost the same with Thymine as they
also have an -O (oxygen) double bonded to the
• Let’s count the number of atoms. 4th carbon. But only contains -H (hydrogen) in the
o Pyrimidine – we have 6 atoms. 2 of which are 5th carbon.
nitrogen. o Thymine – almost the same with Uracil as they
o Purine – we have 9 atoms that comprise the ring. also have an -O (oxygen) double bonded to the
4 of which are nitrogen. 4th carbon. But has a -CH3 (methyl group) in the
5th carbon.
PURINES • Purines
• Is a heterocyclic (consists of more than 1 type of o Adenine – there is an -NH2 (amino group)
atom) aromatic bonded to the 6th carbon
organic o Guanine – has an -NH2 (amino group) bonded to
compound the 2nd carbon AND an -O (oxygen) double
consisting of a bonded to the 6th carbon.
pyrimidine ring
fused to an
imidazole ring

NUCLEIC ACIDS
NUCLEOBASES
• Purines & Pyrimidines Are Heterocyclic Compounds Hydrogen bond involving the amino and carbonyl
• Heterocyclic compounds: cyclic structures that groups: most important mode of interaction between two
contain, in addition to carbon, other (hetero) atoms ( and occasionally three of four) complementary strands
such as nitrogen of nucleic acid.
• Electron delocalization among atoms in the ring → Hydrogen-bonding patterns in the base pairs defined
partial double-bond character → planar purines and by Watson and Crick. Here as elsewhere, hydrogen
pyrimidines → facilitates their close association, or bonds are represented by three blue lines.
“stacking,” that stabilizes double-stranded DNA
• are hydrophobic and relatively insoluble in water at
the near-neutral pH of the cell.
• are hydrophobic and relatively insoluble in water at

the near-neutral pH of the cell.
• Hydrophobic stacking interactions in which two or
more bases are positioned with the planes of their
rings parallel (like a stack of coins)
o The stacking also involves a combination of van
der Waals and dipole-dipole interactions
between the bases.
• Base stacking:
o helps to minimize contact of the bases with water
o very important in stabilizing the three-dimensional
structure of nucleic acids

NUCLEIC ACIDS
NUCLEOSIDES phosphate. And that phosphate binds to the 5’
• Are derivatives of purines and pyrimidines that have carbon of the succeeding sugar.
a sugar linked to a ring nitrogen of a purine or
pyrimidine.
• Numerals with a prime (eg, 2′ or 3′) distinguish atoms
of the sugar from those of the heterocycle.
o The reason why we have 5’ (pron. 5 prime) which
is to differentiate it with the number of the
nucleobases.
o If there is no PRIME SYMBOL (‘), it is
understandable that it comes from the
nucleobase
• The sugar in ribonucleosides is…
o D-ribose
• The sugar in deoxyribonucleosides is…
o 2-deoxy-D-ribose.
• Both sugars are linked to the heterocycle by an a-N-
glycosidic bond (pron. Alpha n glycosidic bond),
almost always to the N-1 of a pyrimidine or to N-9
of a purines
• The N-1 atom of a nucleobase of a pyrimidine forms

a glycosidic bond with the 1’ carbon of the
deoxyribose.
SOURCES OF ATOMS OF PURINES AND
NUCLEOTIDES: PHOSPHORYLATED NUCLEOSIDES
PYRIMIDINES
• Nucleotide: a nucleoside in which a molecule of
phosphoric acid is esterified with an -OH of the
monosaccharide, most commonly either the 3’-OH or
the 5’-OH
• Polymerization leads to nucleic acids.
• Linkage is repeated (3’,5’-phosphodiester bond)
o 3’,5’-phosphodiester bond – (look at the red
arrows in the image below) is made through the
3’ carbon of the adjacent sugar molecule to a

NUCLEIC ACIDS
• Purine (left), pyrimidine (right) • Key Takeaways:
• Where do these molecules come from? o De novo – from scratch, novo = new
o Purine – the sources of atoms of purine are o De novo pyrimidine synthesis
aspartate, formate, Amide N (nitrogen) of ▪ Undergoes a series of chemical changes that
glutamine, CO2, glycine, and another formate. leads to UMP, which can then be used to
o Pyrimidine – the sources of atoms of pyrimidine continue the pathway to become UTP as
are glutamine amid (for one of its nitrogen atom), references in the Nucleotide Biosynthesis
aspartate, HCO3 (bicarbonate) image
BIOSYNTHESIS OF NUCLEOTIDES DE NOVO SYNTHESIS OF PURINE
• The de novo synthesis of purine nucleotide means
using phosphoribose, amino acids, one carbon units
and CO2 as raw materials to synthesize purine
nucleotide from the beginning. (creating from scratch)
• It is the main synthesis pathway of nucleotides.
• Purine synthesis occurs in all tissues. The major site
of purine synthesis is in the liver and, to a limited
extent, in the brain.
o Substrates: Ribose-5-phosphate; glycine;
glutamine; H2O; ATP; CO2; aspartate.
o Products: GMP; AMP; glutamate; fumarate;
IMP: Inosine monophosphate H2O.
AMP: Adenosine monophosphate
GMP: Guanosine monophosphate
ATP: Adenosine triphosphate
GTP: Guanosine triphosphate
UMP: Uridine monophosphate
UTP: Uridine triphosphate
CTP: Cytosine triphosphate
dUMP: Deoxy-uridine monophosphate
dTTP: Deoxy-thymine triphosphate
• PRPP synthetase (Phosphoribosyl pyrophosphate)

is the rate-limiting step in the synthesis of both
purines and pyrimidines.
• Glutamine:PRPP amidotransferase catalyzes the
first-committed step in purine synthesis.
• IMP branch to AMP
o Inhibitor: AMP
o Need for GTP
• IMP branch to GMP
Inhibitor: GMP
o Need for ATP

NUCLEIC ACIDS
PURINE SALVAGE PATHWAY
• Ribose-5-phosphate (as provided by the pentose-

phosphate pathway) is converted into PRPP
(Phosphoribosyl pyrophosphate) by PRPP
synthetase, in a step requiring one ATP.
• In the committed step in the process, an α-amino
group is then added to PRPP from glutamine to form
5-phosphoribosylamine. This reaction is catalyzed by
glutamine PRPP amidinotransferase. • A salvage pathway is a pathway in which nucleotides
• A series of nine reactions results in the formation of are synthesized from intermediates in the degradative
IMP (Inosine 5′-monophosphate). pathway for nucleotides.
• IMP can then be transformed either to GMP by IMP • Bases from degraded nucleic acids can be converted
dehydrogenase, or to AMP by adenylosuccinate back into purine nucleotides via the salvage
synthetase. pathways.
• IMP – very crucial part because it is the starting point • Hypoxanthine can be combined with PRPP (which
for the formation of GMP and AMP (which then acts as the donor of ribose-5 phosphate) to form IMP
becomes Guanine and Adenine) in a reaction catalyzed by Hypoxanthine-guanine
phosphoribosyltransferase (HGPRT).
o HGPRT is important
o A mutation to the HGPRT causes Lesch–Nyhan
syndrome (LNS) which can cause the
accumulation of uric acids.
• IMP can subsequently be transformed into AMP or
GMP via the last few steps of the pathway of de novo
purine synthesis.
• HGPRT also catalyzes the reaction which combines
PRPP with guanine to form GMP.
• Adenine phosphoribosyltransferase converts adenine
and PRPP to form AMP

NUCLEIC ACIDS
PYRIMIDINE SYNTHESIS • Keypoints from the image:
• Biosynthesis of pyrimidine nucleotides can occur by a • Step 1 – CPS-II (pron. carbamoyl phosphate
de novo pathway or by the reutilization of preformed synthetase II) then goes on in a series of processes
pyrimidine bases or ribonucleosides (salvage to reach Step 5
pathway). • Step 5 – orotate with will combine with PRPP to form
• The pyrimidine synthesis is a similar process than that OMP (orotidine 5’-monophosphate)
of purines. • OMP will now undergo decarboxylation to produce
• In the de novo synthesis of pyrimidines, the ring is UMP
synthesized first and then it is attached to a ribose- • UMP is then converted into UTP or CTP
phosphate to for a pyrimidine nucleotide PURINE VS PYRIMIDINE SYNTHESIS
DE NOVO PYRIMIDNE SYNTHESIS
• CO2 and glutamine are combined to form carbamoyl
phosphate. This reaction is catalyzed by carbamoyl
phosphate synthetase II, which is the major regulated
step for this pathway.
• Carbamoyl phosphate is then combined with water
and aspartate before being subsequently
dehydrogenated in a series of reactions to form
orotic acid.
o If it has orotic acid, it is automatically pyrimidine
synthesis
• The ribose-5-phosphate ring is then attached to orotic
acid by orotate phosphoribosyl transferase to form
Orotidine 5′-monophosphate (OMP).
• OMP is decarboxylated to form UMP by OMP
decarboxylase.
• UMP can then be phosphorylated to form UTP.
• UTP can subsequently be converted to CTP with the
addition of an amino group that is donated by
glutamine. The conversion of UTP to CTP is
catalyzed by CTP synthetase.
NUCLEIC ACIDS
LEVELS OF STRUCTURE
• 1°structure: the order of bases on the polynucleotide
sequence; the order of bases specifies the genetic
code
• 2°structure: the three-dimensional conformation of
the polynucleotide backbone
• 3°structure: supercoiling
• 4°structure: interaction between DNA and proteins
2 KINDS
• DNA (deoxyribonucleic acid)
NUCLEIC ACIDS
• RNA (ribonucleic acid)
ASSEMBLED FROM NUCLEOTIDES
• Nitrogenous base
• Five-carbon sugar (pentose)
• Phosphate
NUCLEIC ACIDS
• Polymers of nucleotides
• Joined by phosphodiester bonds
• Phosphate group links the 3’ carbon of a sugar to the
5’ carbon of the next sugar in the chain
• Each strand has a polarity
• 5’ end and 3’ end
• Phosphate group: 5’ end
• Hydroxyl group: 3’ end
• Base sequence is written by convention in the 5’ → 3’
direction

LECTURE | CHRISTIAN JOHN S. CAPIRIG | MIDTERM A.Y. 2021 - 2022
NUCLEIC ACIDS
DEOXYRIBONUCLEIC ACIDS DNA - 2° SRUCTURE
• Deoxyribonucleic acids, DNA: a biopolymer that • Secondary structure: the ordered arrangement of
consists of a backbone of alternating units of 2-deoxy- nucleic acid strands
D-ribose and phosphate o the double helix model of DNA 2° structure was
• the 3’-OH of one 2-deoxy-D-ribose is joined to the 5’- proposed by James Watson and Francis Crick in
OH of the next 2-deoxy-D-ribose by a phosphodiester 1953
bond • Double helix: a type of 2° structure of DNA molecules
• Primary Structure: the sequence of bases along the in which two antiparallel polynucleotide strands are
pentose-phosphodiester backbone of a DNA coiled in a right-handed manner about the same axis
molecule base sequence is read from the 5’ end to o structure based on X-Ray crystallography by
the 3’ end Rosalind Franklin, 1920-1958
• System of notation single letter (A,G,C,U and T)
DNA DIFFERS FROM RNA
• Sugar is 2’-deoxyribose, not ribose.
• In DNA, the two prime carbon is bonded to a
hydrogen while in ribose, the tow prime carbons bind
it to a hydroxyl group.
o Sometimes “d” used to designate “deoxy”
o Writing a DNA strand
▪ an abbreviated notation
▪ even more abbreviated notations d(GACAT)
pdApdCpdGpdT pdACGT
DNA RNA
Molecule is a double Molecule is single-
stranded helix stranded
Sugar is deoxyribose Sugar is ribose
Contains thymine and no Contains uracil, and no
uracil thymine
A=T A =/≠U
G=C G =/≠ C
DNA are usually double-stranded helix. For RNA, they are
usually single-stranded. There are viruses that contain X-RAY DIFFRACTION PATTERN OF DNA FIBERS
single-stranded DNA (Parvo). There are also double-
stranded RNA viruses, Rotavirus.

NUCLEIC ACIDS
The discovery of the structure of DNA was one of the most It is often said that Franklin would have been recognized
important scientific achievements in the last century in by a Nobel prize if only they could be awarded
human history. In fact, the famous double helix is almost posthumously. In fact, it is possible that she could have
synonymous with Watson and Crick, two of the scientists won twice her work on the structure of viruses led to a
who won the Nobel prize for figuring it out. There’s Nobel for a colleague in 1982.
another name, Rosalind Franklin. Her data supported WATSON AND CRICK MODEL OF DNA
Watson and Crick or that she was a plain dressing • DNA consists of two helical DNA chains wound
belligerent scientist which is how Watson actually around the same axis to form a right-handed double
described her in the double killings. Thanks to Franklin’s helix
biographers who investigated her life and interviewed • The hydrophilic backbones of alternating
many people close to her, we know that account is far deoxyribose and phosphate groups are on the outside
from true and her scientific contributions have been vastly of the double helix, facing the surrounding water.
underplayed. • The furanose ring of each deoxyribose is in the C-2’
Rosalind Elsie Franklin was born in London in 1920. She endo conformation.
wanted to be a scientist which wasn’t a common or easy • The purine and pyrimidine bases of both strands are
career path for girls at that time, but she excelled at stacked inside the double helix, with their
science anyway. She won a scholarship to Cambridge to hydrophobic and nearly planar ring structures very
study chemistry where she earned her PhD. She later close together and perpendicular to the long axis.
conducted research on the structure of coal that led to • The offset pairing of the two strands creates a major
better gas masks for the British during World War. In groove and minor groove on the surface of the
1951, she joined King’s College to use X-ray techniques duplex
to study the structure of DNA, then one of the hottest BASE PAIRING
topics in science. Franklin upgraded the X-ray lab and got
o work shining high-energy X-rays on tiny wet crystals of
DNA. However, the academic culture at that time wasn’t
very friendly to women and Franklin was isolated from her
colleagues. She clashed with Maurice Wilkins, a lab mate,
who assumed Franklin had been hired as his assistant.
But Franklin kept working and in 1952, she obtained
Photo51, the most famous X-ray image of DNA. Just
getting the image took 100 hours, the calculations
necessary to analyze it would take a year. Meanwhile, the
American biologist, James Watson, and British physicist,
Francis Crick, were also working on finding DNA
structure. Without Franklin’s knowledge, Wilkins took
Photo51 and showed it to Watson and Crick. Instead of
calculating the exact position of every atom, they did a
quick analysis of Franklin’s data and used that to build a
• Base pairing is complimentary
few potential structures. Eventually, they arrived at the
• A major factor stabilizing the double helix is base
right one. DNA is made of two helicoidal strands—one
pairing by hydrogen bonding between T-A and
opposite the with faces at the center like rungs of a ladder.
between C-G
Watson and Crick published their model in Apil 1953.
Meanwhile, Franklin had finished her calculations come to • T-A base pair comprised of 2 hydrogen bonds
the same conclusion and submitted her own manuscript. • G-C base pair comprised of 3 hydrogen bonds
The journal published the manuscripts together but put COMPLEMENTARY OF STRANDS IN THE DNA
Franklin’s last, making it look like her experiments just DOUBLE HELIX
confirmed Watson and Crick’s breakthrough instead of • the two antiparallel polynucleotide chains of double-
inspiring it. But Franklin had already stopped working on helical DNA are not identical in either base sequence
DNA and died of Cancer in 1958, never knowing that or composition, rather they are complementary to
Watson and Crick had seen photographs. Watson and each other.
Wilkins won the Nobel prize in 1962 for their work on DNA.

NUCLEIC ACIDS
OTHER FORMS OF DNA
CHARGAFF’S RULE
1. The base composition of DNA generally varies from
one species to another.
2. DNA specimens isolated from diﬀerent tissues of the
same species have the same base composition. • B-DNA
3. The base composition of DNA in a given species does o considered the physiological form
not change with an organism’s age, nutritional state, o a right-handed helix, diameter 11Å
or changing environment. o 10 base pairs per turn (34Å) of the helix
4. In all cellular DNAs, regardless of the species, the • A-DNA
number of adenosine residues is equal to the number o a right-handed helix, but thicker than B-DNA
of thymidine residues (that is, A=T), and the number o 11 base pairs per turn of the helix
of guanosine residues is equal to the number of o has not been found in vivo
cytidine residues (G =C). From these relationships it • Z-DNA
follows that the sum of the purine residues equals the o a left-handed double helix
sum of the pyrimidine residues; that is, A + G = T + C. o may play a role in gene expression
SAMPLE PROBLEM
A sample of DNA has 10% C. What is the %T? %A?
%G?

NUCLEIC ACIDS
COMPARISON OF A,B, AND Z FORMS OF DNA INTERMOLECULAR FORCES IN DNA
HELICAL PROPELLER TWISTS

• Bases that are exposed to minor groove contact with
water
• They twist in a “propeller twist” fashion
• Results in:
o less optimal base pair distance
o More optimal base pair stacking (eliminates
presence of water molecules)
• Both A and B-DNA are right-handed helices

• Z-DNA is left-handed
• Z-DNA occurs in nature, usually consists of
alternating purine-pyrimidine bases
• Methylated cytosine found also in Z-DNA
DNA - 3° STRUCTURE
OTHER FEATURES OF DNA
• Tertiary structure: the three-dimensional
• Base stacking
arrangement of all atoms of a nucleic acid; commonly
o bases are hydrophobic (Van der Waals Forces
referred to as supercoiling
of their pi-cloud electrons) and interact by
• Supercoiling: Further coiling and twisting of DNA
hydrophobic interactions
helix.
o in standard B-DNA, each base rotated by 32°
• Circular DNA: a type of double-stranded DNA in
compared to the next and, while this is perfect for
which the 5’ and 3’ ends of each stand are joined by
maximum base pairing, it is not optimal for
phosphodiester bonds
maximum overlap of bases;
DNA TOPOLOGY
o bases exposed to the minor groove come in
• The DNA molecules in human chromosomes are
contact with water
linear
• The DNA molecules in prokaryotes are circular
(refers to the continuity of the DNA strands)
o Negative Supercoils: if the strands are
underwound
o Positive Supercoils: if the strands are
overwound

NUCLEIC ACIDS
DNA SUPERCOILING underwinding should facilitate strand separation over
about 10 bp. However, the hydrogen-bonded base
pairs would generally preclude strand separation over
such a short distance, and the effect becomes
important only for longer DNAs and higher levels of
DNA underwinding.
DNA SUPERCOILING
• Imagine the supercoiling like telephone lines which

results into DNA double helix arrangement, creating
more coiling.
MOST CELLULAR DNA IS UNDERWOUND
• Supercoiling results when DNA is subject to some
form of structural strain.
• In almost every instance, the strain is a result of
underwinding of the DNA double helix in the closed
circle. In other words, the DNA has fewer helical turns
than would be expected for the B-form structure
• Underwinding decreases the total number of helical • Prokaryotic DNA is circular. It can form supercoils.
turns in the DNA relative to the relaxed, B form. • Double helix can be considered to a 2-stranded, right-
• To maintain an underwound state, DNA must be handed coiled rope
either a closed circle or bound to protein. • Can undergo positive/negative supercoiling
EFFECTS OF DNA UNDERWINDING ENZYMES THAT AFFECT THE SUPERCOILING OF
DNA
• Topoisomerases: enzymes that are involved in
changing the supercoiled state of DNA
o Class I: cut the phosphodiester backbone of one
strand, pass the end through, and reseal
o Class II: cut both strands, pass some of the
remaining DNA helix between the cut strands,
and reseal
• DNA gyrase: a bacterial topoisomerase
• (A) A segment of DNA in a closed-circular molecule,

84 bp long, in its relaxed form with eight helical turns.
• (B) Removal of one turn induces structural strain
• (C) The strain is generally accommodated by
formation of a supercoil
o Supercoiling is very important because it allows
easier strand separation for the process of DNA
replication and DNA transcription. It plays a role
in the central dogma.
• (D) DNA underwinding also makes the separation of
strands somewhat easier. In principle, each turn of
NUCLEIC ACIDS
the helix of DNA coils upon itself creating a tighter, more
compact structure.
If one of the strands of a double helix is cut, the DNA loses

its supercoils as the tension dissipates.
However, the other domains remain supercoiled because

they are constrained at their bases by the anchoring
proteins which prevent rotation. TO understand
supercoiling, consider a completely relaxed circular piece
of DNA—an unstressed DNA molecule naturally forms a
helix with about 10 base pairs per turn.
Video about Supercoiling

In a remarkable experiment, a
cell of e-coli was lysed releasing
its chromosome for electron
microscopy.
What spewed out of this single cell
was a strand of DNA 1500 times
longer than e-coli itself. How could
this enormous molecule fit into a
single cell? Inside a bacterial cell,
the DNA is much more tidy. The
DNA is compacted into a structure
called the nucleoid.
The nucleoid consists of

Consider cutting a strand underwinding a turn and then
DNA arranged in tightly
resealing the DNA.
wound loops with
boundaries of the loop
domains defined by
histone-like anchoring
proteins. The DNA in the
The DNA is now underwound and has one too few helical
loops is super cool that is
turns. The DNA will naturally attempt to return to the same

NUCLEIC ACIDS
number of turns as before. But, considering the DNA has Topoisomerase one cleaves one
been resealed in this underwound form, it must create a strand of a double helix holds on to
supercoil in the opposite direction to relieve the stress. both ends and passes the other
intact strand through the break.
After which, it relegates the strand.
The topoisomerase comes off the
DNA leaving the circular molecule
with four supercoils instead of five.
An example of a type 2
topoisomerase is DNA gyrase
whose function is to introduce
negative supercoils in double-
Negative supercoils relieve torsional strain of underwound stranded DNA rather than to
DNA while positive supercoils relieve overwound DNA. remove them. The active gyrase
This circular double-stranded DNA has complex is a tetramer composed
five negative supercoils. If supercoils were of two GyrA and GyrB proteins. GyrB grabs one section
removed, the DNA would be revealed to of the double stranded DNA the GyrA introduces a double
be underwound by about five turns. -stranded DNA then GyrA introduces a double-strand
break in the DNA and becomes covalently attached to the
breaks as it holds the two ends apart.
Adding a negative supercoil
to DNA requires energy
which is gained through
ATP hydrolysis. GyrA which
is an ATPase uses the
The torsional stress energy of ATP hydrolysis to
in the underwound pass the intact double-
DNA is relieved by stranded section through double-strand break.
rewinding and at the
same time creating
negative supercoils.
How does a DNA achieve a supercoiled
state? A bacterial cell produces enzymes
that can twist DNA into supercoils and relieve
supercoils. Enzymes that change DNA GyrB rejoins the cleave DNA and opens at the other end
supercoiling are topoisomerases because to release the newly sealed strand. The DNA molecule
they change the topology of DNA. Type 1 contains one negative supercoil. To maintain proper DNA
topoisomerases cleave only one strand of a supercoiling levels, a cell must delicately balance the
double helix while type enzymes cleave both activities of the two types of topoisomerases. The
strands. nucleoids of bacteria and most archaea, as well as the
nuclear DNA of eukaryotes, are kept negatively
Topoisomerase 1 binds to supercoiled because the DNA is underwound the two
DNA and opens two strands strands of negatively supercoiled DNA are easier to
loosening the double helix. separate than positively supercoiled DNA which is
This loss of a turn in the important for transcription enzymes like RNA polymerase
double helix allows the DNA that must separate stands of DNA to make RNA.
to convert from five
supercoils to four.

NUCLEIC ACIDS
Some archaeal CHROMATIN: CHROMOSOMAL MATERIAL IN THE
species living in NUCLEI OF CELLS OF EUKARYOTIC ORGANISMS
acid at high • Chromatin
temperature consists of:
have a challenge o very long
of keeping their double-stranded DNA
DNA from denaturing that is separating into single- (dsDNA) molecules
stranded DNA. Their DNA is overwhelmed resulting in o a nearly equal
positive supercoils. It is proposed that the extra turns in mass of small basic
the DNA tighten the coil which would require more energy proteins termed
in the form of heat to separate the strands. histones
Moreover, it makes DNA accessible to the polymerase o nonhistone
enzymes especially in gene expression. proteins (most of which
SUPERCOILING IN EUKARYOTES are acidic and larger
• Eukaryotic DNA is complexed with a number of than histones)
proteins, especially with basic proteins that have o small quantity of
abundant positively charged side chains at RNA
physiological (neutral) pH.
• Electrostatic attraction between the negatively
charged phosphate groups on the DNA and the
positively charged groups on the proteins favors
the formation of complexes of this sort. The resulting
material is called chromatin.
• Topological changes induced by supercoiling must be
accommodated by the histone-protein component of
chromatin The mechanisms of life stimulated through
ORGANIZATION OF EUKARYOTIC CHROMOSOMES supercomputers.
We are inside a cell in a special part called nucleus. We

see some DNA which is encoded with genetic information
including genes, the blueprint of cells. The motions of a
vast number of water molecules have been calculated to
accurately stimulate the behavior of molecules inside the
DNA is like a long string composed of a double-stranded
helix folded into very compact structures in the cell
nucleus.
The center of each of

these structures is
composed of a protein
called histone. Each
center consists of
eight protein, two
copies each of four
types of histones.

NUCLEIC ACIDS
The tumor suppressor protein called p53 is about to bind
to the DNA. When the DNA is unwrapped, p53 can move
along the DNA and read its genetic information.
Chemical modifications such as acetylation and
differences in chromatin affect the reading of the
genetic information. This allows various cells and tissues
such as red blood cells and nerves to be generated from
the same DNA. The study of mechanisms by which genes
are turned on and off without changes in the underlying
DNA sequence is called epigenetics. Studies using
Researchers have been working hard to understand their supercomputers to stimulate the motions and shapes of
roles. This structural unit formed by DNA wrapped around molecules will contribute to the development of new drugs
the histone proteins is called a nucleosome. By treating and research on regenerative medicine.
multiple atoms as one single particle, we can increase the HISTONES: MOST ABUNDANT CHROMATIN
number of phenomena in our simulation. Human DNA PROTEINS
measures about two meters in length but it can fit in a tiny
nucleus of only 10 micrometers in diameter by forming a
fiber of packed nucleosomes called chromatin. For the
genetic information to be read, the DNA has to be
unwrapped from the histones. K computer has made it
possible for the first time ever to calculate the force
required at the atomic level to unwrap the DNA. It is also
known that changes in histone state have unstructured
flexible regions. These are called histone tails. When
histone tails undergo modification such as acetylation,
the structure of the nucleosome changes. For instance, • Histone: a protein, particularly rich in the basic
we know that acetylation specific parts of the histone tails amino acids Lys and Arg (positive in neutral pH);
loosen the chromatin structure, making the nucleosomes found associated with eukaryotic DNA
less tightly packed. o five main types: H1, H2A, H2B, H3, H4
o the DNA is tightly bound to all the types of histone
except H1
• Nucleosome: DNA wound around an octameric
complex of histone molecules.
• H1 histones are the ones least tightly bound to
chromatin and are, therefore, easily removed with a
salt solution, after which chromatin becomes more
soluble.
The DNA has to unwrap from the histone proteins to allow • The organizational unit of this soluble chromatin is the
proteins to read its genetic information. nucleosome.
• Nucleosomes contain four major types of histones:
H2A, H2B, H3, and H4.
HISTONES
• The carboxyl terminal two-thirds of the histone
molecules are hydrophobic
• The amino terminal thirds are particularly rich in
basic amino acids
• These four core histones are subject to at least six
types of covalent modification or posttranslational
modifications (PTMs):
▪ These histones can be chemically modified to
change the behavior of the binding of the

NUCLEIC ACIDS
DNA or the coiling of the DNA to that histone are thought to extend outside of this structure and are
protein and it can affect on how several available for regulatory PTMs
enzymes and proteins that will bind to or • The (H3–H4)2 tetramer itself can confer nucleosome-
transcription factors that will bind to the DNA like properties on DNA and thus has a central role in
to express a certain gene. the formation of the nucleosome.
o Acetylation • The addition of two H2A–H2B dimers stabilizes the
o Methylation primary particle and firmly binds two additional
o Phosphorylation half-turns of DNA previously bound only loosely to
o ADP-ribosylation the (H3–H4)2.
o Monoubiquitylation • 1.75 superhelical turns of DNA are wrapped around
o Sumoylation the surface of the histone octamer, protecting 145 to
150 bp of DNA and forming the nucleosome core
particle
• In chromatin, core particles are separated by a
roughly 30-bp region of DNA termed “linker.”
• Most of the DNA is in a repeating series of these
structures, giving chromatin a repeating “beads-on-a-
string” appearance when examined by electron
microscopy
• The histones interact with each other in very specific
ways.
• H3 and H4 form a tetramer containing two molecules
of each (H3–H4)2,
• H2A and H2B form dimers (H2A– H2B).
• Under physiologic conditions, these histone
oligomers associate to form the histone octamer of
the composition (H3–H4)2–(H2A–H2B)2.
NUCLEOSOMES
• In the nucleosome, the DNA is supercoiled in a left-

handed helix over the surface of the disk-shaped
histone octamer
Linker DNA are strands of DNA that are not associated

to any histone proteins.
HISTONE TAILS
• The majority of core histone proteins interact with the
DNA on the inside of the supercoil without protruding,
although the amino terminal tails of all the histones

NUCLEIC ACIDS
This shows the importance of histone tail. Most likely, they • 30-nm chromatin fiber: 10-nm fibril is further
consist of a lot of arginine and lysine. K and R are sites supercoiled with six or seven nucleosomes per turn to
where in acetylation, methylation, phosphorylation, and form
ubiquitination can take place. This process is what we call SUPERCOILING IN EUKARYOTIC DNA
post-translational modification. • Chromatin: DNA molecules wound around particles
NUCLEOSOME of histones in a beadlike structure
• Topological changes induced by supercoiling
accommodated by histone-protein component of
chromatin.
DENATURATION OF DNA
• DNA is wrapped around the surface of a protein

cylinder consisting of two each of histones H2A, H2B,
H3, and H4 that form the histone octamer.
• The ~145 bp of DNA, consisting of 1.75 superhelical
turns, are in contact with the histone octamer.
• The position of histone H1, when it is present, is
indicated by the dashed outline at the bottom of the • Denaturation: disruption of 2° structure
figure. o most commonly by heat denaturation (melting)
• Note that histone H1 interacts with DNA as it enters o as strands separate, absorbance at 260 nm
and exits the nucleosome increases
COMPACTION OF DNA IN A EUKARYOTIC o increase is called hyperchromicity
CHROMOSOME o midpoint of transition (melting) curve = Tm
• Nucleosomes Are Packed into Successively Higher- o the higher the % G-C, the higher the Tm
Order Structures ▪ if you have a lot of G-C, you have three
hydrogen bonds compared to an 80. You
have a higher temperature needed to
separate the strands.
o renaturation is possible on slow cooling
• Double helix unwinds when DNA is denatured
• Can be re-formed with slow cooling and annealing
HIGHER ORDER STRUCTURE TO PROVIDE

COMPACTION
• Electron microscopy of chromatin reveals two higher
orders of structure—the 10-nm fibril and the 30-nm
chromatin fiber PRINCIPAL KINDS OF RNA
• 10-nm fibril: consists of nucleosomes arranged with RNA
their edges separated by a small distance (30 bp of • consist of long, unbranched chains of nucleotides
linker DNA) with their flat faces parallel to the fibril axis joined by phosphodiester bonds between the 3’-OH
of one pentose and the 5’-OH of the next
NUCLEIC ACIDS
• the pentose unit is β-D-ribose (it is 2-deoxy-D-ribose
in DNA)
• the pyrimidine bases are uracil and cytosine (they
are thymine and cytosine in DNA)
• in general, RNA is single stranded (DNA is double
stranded)
CLASSIFICATION OF RNA
mRNA – gene expression, specifically for translation

non-coding RNA is divided into a housekeeping non-
coding RNA and regulatory encoding RNA.
INFORMATION TRANSFER IN CELLS
• Information encoded in the nucleotide sequence of
DNA is transcribed through RNA synthesis
• Sequence then dictated by DNA sequence
• Central dogma of biology
Transcription – an enzyme will read a certain segment of

the DNA to make this mRNA. mRNA will be read by the
ribosome to form protein. That protein will exert its
function, whether in structural, chemical or enzymatic
function.

NUCLEIC ACIDS
mRNA IN TRANSCRIPTION rRNA
• Ribosomal RNA, rRNA: a ribonucleic acid found in
ribosomes, the site of protein synthesis
o only a few types of rRNA exist in cells
o ribosomes consist of 60 to 65% rRNA and 35 to
40% protein
o in both prokaryotes and eukaryotes, ribosomes
consist of two subunits, one larger than the other
o analyzed by analytical ultracentrifugation
o particles characterized by sedimentation
coefficients, expressed in Svedberg units (S)
mRNA
• Messenger RNA, mRNA: a ribonucleic acid that
carries coded genetic information from DNA to
ribosomes for the synthesis of proteins
o present in cells in relatively small amounts and
very short-lived
RNA o single stranded
o biosynthesis is directed by information encoded
on DNA
o a complementary strand of mRNA is synthesized
along one strand of an unwound DNA, starting
from the 3’ end
snRNA
• Small nuclear RNA (snRNA) is a recently discovered
RNA
• Found in nucleus of eukaryotes
• Small (100-200 nucleotides long)
tRNA
• Forms complexes with protein and form small nuclear
• Transfer RNA, tRNA: ribonucleoprotein particles (snRNPs)
o the smallest kind of the three RNAs
• snRNPs help with processing of initial mRNA
o a single-stranded polynucleotide chain between
transcribed from DNA
73-94 nucleotide residues
miRNA
o carries an amino acid at its 3’ end
• A microRNA (abbreviated miRNA) is a small single-
o intramolecular hydrogen bonding occurs in tRNA
stranded non-coding RNA molecule (containing
about 22 nucleotides) found in plants, animals and
some viruses, that functions in RNA silencing and
post-transcriptional regulation of gene
expression.
• miRNAs function via base-pairing with
complementary sequences within mRNA molecules.
• As a result, these mRNA molecules are silenced, by
one or more of the following processes: (1) cleavage
of the mRNA strand into two pieces, (2)
destabilization of the mRNA through shortening of its
poly(A) tail, and (3) less efficient translation of the
mRNA into proteins by ribosomes.

NUCLEIC ACIDS
REVIEW ARTICLE
The Role of MicroRNAs in human cancer
Yong Peng and Carlo M. Croce
MicroRNAs (miRNAs) are dangerous, small non-coding

RNAs that function in regulation in gene regulation of
gene expression. Compelling evidences have
demonstrated that miRNA expression is dysregulated in
human cancer through various mechanisms, including
amplification or deletion of miRNA genes, abnormal
transcriptional control of miRNAs, dysregulated
epigenetic changes and defects in the miRNA biogenesis
machinery. MiRNAs may function as either oncogenes or
tumor suppressors under certain conditions. The
dysregulated miRNAs have been shown to affect
hallmarks of cancer, including sustaining proliferative
signaling, evading growth suppressors, resisting cell
death, activating invasion and metastasis, and including
angiogenesis. An increasing number of studies have been
identified miRNAs as potential biomarkers for human
cancer diagnosis, prognosis and therapeutic targets or
tools which needs further investigation and validation. In
this review, we focus on how miRNAs regulate the
development of human tumors by acting tumor
suppressors and oncogenes.
Signal Transduction and Targeted Therapy (2016) 1,

15004; doc:10.1038/sigtrans.2015.4;published online 28
January 2016
USE OF miRNA

LECTURE | PROF. CHRISTIAN JOHN S. CAPIRIG, MD | MIDTERM A.Y. 2021 - 2022
THE CENTRAL DOGMA OF MOLECULAR BIOLOGY
OUTLINE
I. DNA Replication
II. Transcription
III. Translation
CENTRAL DOGMA OF MOLECULAR BIOLOGY
• “DNA makes RNA, and RNA makes protein”
DNA REPLICATION
HOW DOES DNA PRODUCE MULTIPLE COPIES
EFFICIENTLY?
• Reproduction is fundamental to all living systems
• Regardless of the reproductive mechanism (asexual
or sexual) a method must exist to transfer genetic
material from one generation to the next.
• DNA must be copied (replicated) in a manner that
minimizes mistakes.
• Damage to DNA must be repaired to prevent that
damage from being transferred to the next
generation.
DNA REPLICATION
• Watson & Crick (1953) - proposed DNA structure &
suggested how it might "self-duplicate"
• “It has not escaped our notice that the specific pairing
we have postulated immediately suggests a possible
copying mechanism for the genetic material.”
o A. Suggested that replication occurred by gradual
double helix strand separation via successive
breakage of H bonds, much like the separation
of the two halves of a zipper
o B. Since each strand is complementary to the
other, each has the information needed to
construct the other; once separated, each strand
can serve as template to direct the formation of
the other strand
• Possible types of DNA replication
o 1. Semiconservative - daughter duplex made of
one parental & one newly synthesized strand
o 2. Conservative - 2 original strands stay together
after serving as templates for 2 new strands that
also stay together; one contains only "old" DNA,
the other only "new" DNA
o 3. Dispersive – integrity of both parental strands
disrupted; new duplex strands made of old & new
DNA; neither the parental strands nor the
parental duplex is preserved

• Semiconservative replication
• Conservative replication
DNA REPLICATION
• Semiconservative nature of replication -Watson &
Crick predicted that new DNA should consist of
one old strand (from parental duplex) & one newly
synthesized chain
o As with any scientific prediction (a hypothesis),
scientists have an obligation to TEST it • Dispersive replication
o If your experiments refute the hypothesis, you
must generate a new one
o Other scientists tested Watson and Crick’s
prediction in 1957
• Matthew Meselson & Franklin Stahl (1957, Caltech)
grew bacteria in media with 15NH4Cl as sole nitrogen
source for many generations; DNA bases contain
"heavy" nitrogen
o 1. Wash out 15NH4Cl (ammonium chloride); put
• What Meselson and Stahl observed…
bacteria in 14NH4Cl ("light"); remove samples at
o 4. Density of DNA decreases until one generation
intervals over several generations; use 14N (light)
time when it is halfway between the density of
& 15N (heavy) to distinguish between newly
totally heavy & totally light DNA; it is a hybrid —
synthesized & parental strands, respectively
half new & half old
o 2. Extract DNA & subject it to CsCl equilibrium
o 5. After 2 generation times, half of the DNA is
density-gradient centrifugation to find buoyant
totally light & half is hybrid (half light, half heavy)
density
o 6. While semiconservative replication continues,
o 3. Mix DNA with concentrated CsCl (cesium
original heavy parental strands remain intact &
chloride) solution, centrifuge until double-
present in hybrid DNA molecules, but they
stranded DNAs reach equilibrium according to
occupy a smaller & smaller percentage of total
their density; density of DNA is directly related to
DNA
percentage of 14N or 15N it contains
o 7. With time, the vast majority of DNA present is
DNA REPLICATION
fully light with 2 light strands
• Predictions of the Meselson-Stahl Experiment

MESELSON AND STAHL’S EXPERIMENT
DNA REPLICATION IS SEMI-CONSERVATIVE

Each replicated DNA molecule would consist of one “old”
and one “new” strand. The old pair will separate to have a
new partner.
THE MESELSON-STAHL EXPERIMENT

Provided strong evidence of semiconservative replication
DNA containing a 50–50 mixture of 14N and 15N appears

at a position halfway between the two bands. In the actual
experiment, this 50– 50 hybrid DNA was observed after
one generation, a result to be expected with
semiconservative replication. After two generations in the
lighter medium, half of the DNA in the cells should be the
50–50 hybrid and half should be the lighter 14N DNA
SAMPLE PROBLEM
• Consider the experiment conducted by Meselson and
Stahl in which they used 14N and 15N in cultures of
E. coli and equilibrium density gradient centrifugation.
Predict the type of replication that occurred given the
following results:
one by one to the 3’ end of fragment 3. After the last
addition, the backbone is left with a free 3’ end.
7. DNA ligase joins the 3’ end of fragment 2 to 5’ end of
fragment 1.
• Main point – there is a leading and lagging strand.
o Leading strand – One strand that will proceed
with DNA replication continuously in the same
direction of the unwinding of DNA.
o Lagging strand – would have an opposite
• 15N tends to be at the lower part because it is heavy direction of DNA synthesis.
• Reannealing – going back together
ANSWER • Okazaki fragment – only present in the lagging
• Conservative Replication. In this model, the entire strand
molecule serves as a template. After one round of
replication, some molecules will consist entirely of
15N, and others will consist entirely of 14N; so two
bands should be present. Subsequent rounds of

replication will increase the fraction of DNA consisting
entirely of new 14N; thus, the upper band will get
darker. However, the original DNA with 15N will
remain, and so two bands will be present.
ANALYSIS QUESTION
• In the Meselson–Stahl experiment, which of the three
modes of replication could be ruled out after one
round of replication? After two rounds?
• Everything starts with the origin of replication.

1. Helicase (enzyme) unwinds/unzips the parental DNA
helix.
2. Molecules of single-strand binding protein stabilize
the unwound template strands.
• DNA polymerases – catalyze the synthesis of new
3. The leading strand is synthesized continuously in the
DNA by adding nucleotides to a preexisting chain.
5’ to 3’ direction by DNA pol III (DNA polymerase III).
4. Primase begins synthesis of the RNA primer for the
fifth Okazaki fragment.
5. DNA pol II is completing synthesis of fragment 4.
When it reaches the RNA primer on fragment 3, it will
detach and begin adding DNA nucleotides to the 3’
end of the fragment 5 primer in the replication fork.
6. DNA pol I removes the primer from the 5’ end of
fragment 2, replacing it with DNA nucleotides added
• DNA polymerase catalyzes the addition of nucleotide SYNTHESIS OF THE LAGGING STRAND
to the 3’ end of a growing DNA strand, with the • Synthesis of the strand is away from the replication
release of 2 phosphates. fork
INCORPORATION OF NUCLEOTIDE INTO A DNA • DNA polymerase III – forms Okazaki Fragments in
STRAND 5’ to 3’ direction
• This is the mechanism at which polymerases add • DNA polymerase I – replaces RNA nucleotides of the
nucleotides to the existing primer. adjacent primer with DNA nucleotides
• The 3'-hydroxyl group at the end of the growing DNA • DNA ligase – joins the sugar-phosphate backbones
chain is a nucleophile. of all Okazaki fragments into a continuous DNA
• It attacks at the phosphorus adjacent to the sugar in strand.
the nucleotide, which is added to the growing chain. • Replication fork – the portion where the DNA is
• Pyrophosphate is eliminated, and a new being separated.
phosphodiester bond is formed.
PROOFREADING AND REPAIRING DNA

1. Teams of enzymes detect and repair damaged DNA, nonreplicated segments; each replication fork
such as this thymine dimer (often caused by corresponds to a site where the:
ultraviolet radiation), which distorts the DNA ▪ 1. Parental double helix is undergoing strand
molecule. separation, and
2. A nuclease enzyme cuts the damaged DNA strand at ▪ 2. Nucleotides are being incorporated into the
two points, and the damaged section is removed. newly synthesized complementary strands
3. Repair synthesis by a DNA polymerase fills in the o C. The 2 replication forks move in opposite
missing nucleotides. directions & meet at point across the circle from
4. DNA ligase seals the free end of the new DNA to the origin, where replication terminates —> newly
old DNA, making the strand complete. replicated duplexes detach from one another &
• DNA polymerases proofread each nucleotide go to different cells
against its template as soon as it is covalently bonded
to the growing strand. Upon finding an incorrectly
paired nucleotide, the polymerase removes the
nucleotide and then resumes synthesis.
• A segment of the strand containing the damage is cut
out (excised) by a DNA-cutting enzyme— a
nuclease—and the resulting gap is then filled in with
nucleotides, using the undamaged strand as a
template.
o Exonucleases
▪ Degrade DNA from free 3 ′ -hydroxyl or 5 ′ -
phosphate ends
▪ Works only in linearized DNA
o Endonucleases
▪ break the sugar-phosphate backbone of DNA
(internally)
▪ Example: restriction enzymes, CRISPR Cas-
9
REPLICATION IN BACTERIAL CELLS: THE OVERALL
PROCESS
• Genetic & biochemical approaches have revealed at
least 30 proteins needed for E. coli replication
• Several approaches have driven progress in
understanding prokaryotic replication (eukaryotes are
more complicated):
o A. Replication begins at a specific site on
bacterial chromosome (origin)
▪ 1. It starts at specific sequence (called oriC in
E. coli) on a bacterial chromosome
• oriC = origin of replication in E. coli
• Separation of the strands of a circular, helical DNA
▪ 2. Many proteins bind at oriC to initiate
duplex or a giant, linear eukaryotic chromosome
replication; bacterial replication origin
poses major topological problems
analogous to promoter for transcription; both
o A. Analogy: take two-stranded helical rope &
bind sequence-specific, DNA-binding
place linear piece of it on the ground
proteins to start process at specific site
▪ 1. Grab both strands at one end & begin
▪ 3. Replication moves out from origin in both
pulling them apart (just like replicating DNA
(opposite) directions (bidirectional)
does)
o B. Replication forks are sites where the pair of
replicated segments come together & join the

▪ 2. Strand separation of a double helix also Uncoiling of the [DNA] structure is the
involves the process of unwinding the initiative step for replication, transcription and
structure repair of the DNA. Thus, prolonged inhibition
▪ 3. A rope is free to rotate around its axis so will eventually lead to the death of the cell.”
separation of strands at one end is • DNA polymerase -synthesizes new DNA strands
accompanied by rotation of the entire fiber as • Template DNA must meet certain structural
it resists the development of tension requirements to promote dNTP incorporation
• If second end is fixed, strand separation generates (polymerization)
increasing torsional stress in rope; unseparated o 1. Intact, linear, double-stranded DNA did not
portion is wound more tightly stimulate incorporation –not surprising since
o 1. Separation of 2 strands of circular DNA or strands of helix had to be separated for replication
linear DNA that is not free to rotate (like to occur
eukaryotic chromosome) is analogous to o 2. Single-stranded, circular DNAs also cannot
attaching one end of linear molecule to a wall serve as template
o 2. Tension cannot be relieved by rotation of the o 3. In contrast, partially double-stranded DNA
entire molecule works well & yields immediate nucleotide
o 3. Unlike a rope, which can become tightly incorporation
overwound, an overwound DNA molecule
becomes positively supercoiled
o 4. Thus, replication fork movement generates
positive supercoils in unreplicated portion of DNA
ahead of fork
• Cells contain enzymes (topoisomerases) that can

change state of DNA supercoiling - one is DNA
gyrase
o 1. DNA gyrase travels along DNA ahead of fork
removing positive supercoils & changing
positively supercoiled DNA into negatively
supercoiled DNA
o 2. It cleaves both DNA duplex strands, passing a
• It was soon discovered that a single-stranded DNA
segment of DNA through the double-stranded
circle does not serve as DNA polymerase template
break to the other side & then seals the cuts
because the enzyme cannot initiate DNA strand
o 3. Eukaryotic cells possess similar enzymes that
formation
carry out this required function
o 1. Polymerase can only add nucleotides to 3'-OH
• Why bother knowing this stuff? end of an existing strand; a primer is necessary •
o Cipro is an antibiotic that happens to be effective DNA polymerase can only work in a 5’ –3’
against anthrax bacteria, as well as many other direction
types of bacteria. It is helpful in treating bacterial o 2. All prokaryotic & eukaryotic DNA polymerases
infections that cause everything from bronchitis to have these same 2 basic requirements:
gonorrhea. ▪ 1 -a primer strand to which nucleotides can
o According to the Bayer’s site, Cipro works in the be added &
following way: ▪ 2 -a template DNA strand to copy
▪ “...it inhibits bacterial nuclear DNA synthesis,
so that bacteria rapidly die. The target is the
enzyme DNA gyrase (topoisomerase II)…,

o 1. Polymerases building both strands move in 3'
to 5' direction along the template; both make a
chain that grows from its 5'-phosphate terminus •
o 2. One DNA strand grows toward replication fork;
the other away from the fork; both grow 5'—>3' •
o How is this done?
▪ 1. The strand growing toward the fork grows
continuously in 5'—>3' direction (leading
strand) as the replication fork advances
▪ 2. The strand growing away from fork
(lagging strand) grows discontinuously as
Okazaki fragments
▪ 3. Both strands are probably made at same
time so the leading & lagging terms may not
be as appropriate as was thought when they
were first coined
• DNA polymerase findings raised 2 important

questions
o 1. Watson & Crick originally predicted that one
strand at replication fork would be made in 5'—
>3' direction, while the other was made in 3'—>5'
direction —how does a 5'—>3' onlypolymerase
do this?
o 2. If it cannot initiate strands on its own, how does
DNA polymerase initiate synthesis of a new
strand in the cell?
• Strand initiation is done by an enzyme that makes a

short RNA primer, a distinct type of RNA polymerase,
called primase, that constructs a short primer made
of RNA, not DNA
• Leading strand synthesis is initiated at the replication
origin by a primase molecule
• Short RNAs made by primaseat 5' end of leading
strand & the 5' end of each Okazaki fragment serve
as the required primer for synthesis of DNA by a DNA
polymerase
• The RNA primers are subsequently removed & the
• Semidiscontinuous replication-all DNA resulting gap in the strand is filled with DNA & then
polymerases lay down nucleotides in 5'—>3' direction sealed by DNA ligase
& move along template in 3'—>5' direction; REVIEW FROM THE LAST TIME

• Be able to describe the Meselson-Stahl experiment
with regard to purpose, method, results, and
conclusions.
• Be able to describe the general process of bacterial
DNA replication.
• Be able to describe the source and consequences of
the torsional stress induced by movement of the
replication fork.
• Topoisomerases are molecules that can relieve that
stress.
• DNA polymerases have three major features:
o They must have a template, they must have a
primer, and they must polymerize in a 5’-3’
direction.
• Be able to describe how these features affect the
behavior of the replication fork.
• DNA polymerase findings raised 2 important
questions
o 1. Watson & Crick originally predicted that one
strand at replication fork would be made in 5'—
>3' direction, while the other was made in 3'—>5'
direction —how does a 5'—>3' onlypolymerase
do this?
o 2. If it cannot initiate strands on its own, how does
DNA polymerase initiate synthesis of a new
strand in the cell?
• Strand initiation is done by an enzyme that makes a
REPLICATION IN BACTERIAL CELLS: ALL THE
short RNA primer, a distinct type of RNA polymerase,
PLAYERS WORK TOGETHER AT THE REPLICATION
called primase, that constructs a short primer made
FORK
of RNA, not DNA
• DNA helicase (DNA unwinding enzyme) -unwinds
• Leading strand synthesis is initiated at the replication
DNA in reaction using energy from ATP hydrolysis to
origin by a primase molecule
break H bonds holding 2 strands together, thus
• Short RNAs made by primaseat 5' end of leading
exposing the single-stranded DNA template
strand & the 5' end of each Okazaki fragment serve
o E. coli has at least 12 different helicases
as the required primer for synthesis of DNA by a DNA
o DnaBhelicase, the product of the dnaBgene, is
polymerase
the major unwinding machine during replication;
• ZThe RNA primers are subsequently removed & the
it consists of 6 subunits arranged to form a ring-
resulting gap in the strand is filled with DNA & then
shaped protein that encircles a single DNA strand
sealed by DNA ligase
▪ 1. moves in 5'—> 3' direction along lagging-
strand template, unwinding helix as it
proceeds
▪ 2. DNA unwinding by helicase is aided by
attachment of SSB proteins to separated
DNA strands
▪ 3. SSBs bind selectively to single-stranded
DNA -keep it extended & prevent it from
being rewound

• Primase enzyme initiates synthesis of each Okazaki

fragment helicase associates transiently with primase
in bacteria forming primosome
o A. Helicase moves along the lagging strand
template without being released from the
template strand during the lifetime of the
replication fork
o B. As helicase moves along the lagging-strand o 4. Both polymerases can then move together as
template opening duplex strands, primase part of a single replicative complex (replisome)
periodically binds to helicase & synthesizes short without violating the 5'—>3' directionality rule for
RNA primers that begin formation of Okazaki synthesis of a DNA strand
fragment o 5. Once the polymerase assembling the lagging
▪ Primers are subsequently extended as DNA strand reaches the 5' end of the Okazaki fragment
by a DNA polymerase made during the previous round, the lagging-
• The trombone model strand template is released
• DNA polymerase on the lagging strand moves from o 6. The polymerase then begins work at the 3' end
one completed fragment on the template to a site of the next RNA primer toward the fork
closer to replication fork
o 1. It hitches a ride with the DNA polymerase that
is moving that way along the leading strand
template
o 2. The 2 polymerases are part of a single protein
complex even though they move in opposite
directions with respect to each of their templates
o 3. Replication of both strands by the 2 tethered
polymerases can be done by having the DNA of
the lagging strand looped back on itself so it has
same orientation as the leading strand template
REPLICATION IN BACTERIAL CELLS: MULTIPLE

POLYMERASES
• A. DNA polymerase I –
o mostly involved in DNA repair to correct damaged
DNA sections;
o consists of a single subunit;
o removes RNA primers at 5' Okazaki fragment end
& replaces the RNA with DNA
• B. DNA polymerase II – o 1. DNA polymerase discriminates among 4
o function as yet uncertain; bacterial mutants have different precursors as they move in & out of
been isolated & have no evident deficiency active site
• C. DNA polymerase III (replicase) – o 2. Only one of them forms a proper geometric fit
o acts in DNA strand formation during replication in with the template, producing an A-T or G-C base
E. coli; pair that fits in the enzyme active site
o part of a large complex called DNA polymerase III o 3. If incoming nucleotide is correct, a
holoenzyme or replisome, a large replication conformational change occurs in which the
machine polymerase rotates gripping the incoming
REPLICATION IN BACTERIAL CELLS: nucleotide
EXONUCLEASE ACTIVITY o 4. If the newly formed base pair exhibits improper
• DNA polymerases also degrade nucleic acid geometry, the active site cannot achieve the
polymers (exonuclease activity); all bacterial conformation required for catalysis & the incorrect
polymerases possess exonuclease activity; a nucleotide is not incorporated
seeming contradiction o 5. In contrast, if the base pair exhibits proper
• Exonucleases are divided into 5'—>3' & 3'—>5' geometry, the incoming nucleotide is covalently
exonucleases, depending on the direction in which linked to the end of the growing strand
the strand is degraded o Despite this mechanism, incorrect pairings
• DNA polymerase I has both 5'—>3' & 3'—>5' sometimes occur (~1 time for every 105–
exonuclease activities, along with its polymerase 106nucleotides)
activity • Polymerase's 3'—>5' exonuclease activity takes care
o DNA polymerase I 5'—>3' exonuclease activity of mismatched bases
degrades both RNA & DNA (unusual, since • This can happen right after misincorporation or later
usually exonucleases are specific for & degrade on (mismatch repair)
one or the other) • Thus, there are three mechanisms to ensure accurate
▪ 1. Exonuclease removes the RNA primer laid replication
down by the primase at Okazaki fragment 5' o 1. Accurate selection of nucleotides
end; polymerase simultaneously fills in the o 2. Immediate proofreading
resulting gap with deoxyribonucleotides o 3. Postreplicative mismatch repair
▪ 2. DNA ligase covalently joins the last REPLICATION IN EUKARYOTES: INITIATION
deoxyribonucleotide added during RNA • Higher organisms' cells have much more DNA than
primer digestion & the 5' end of the previously bacteria & incorporate DNA at much slower rates;
synthesized & adjacent DNA fragment thus, they initiate replication at many sites rather than
REPLICATION IN BACTERIAL CELLS: ENSURING just one as with the circular chromosome of E. coli
FIDELITY
• Organism survival depends on accurate genome
duplication
o A mistake in DNA replication results in a
permanent mutation in the genetic material & the
possible elimination of that cell's progeny
▪ 1. In E. coli, the chance of incorrect
nucleotide incorporation is <10-9; fewer than
1 out of 1 billion nucleotides
▪ 2. The E. coli genome contains ~4 x 106
• Initiation of DNA synthesis in a givencell is subject to
nucleotide pairs, Thus, this error rate
regulation
corresponds <1 nucleotide alteration for
o 1. Replicons close to each other on a given
every 100 replication cycles
chromosome tend to replicate simultaneously
• Infidelity is detected based on the chemical
o 2. In mammals, timing of replication in a
conformation of nucleotide pairs
chromosomal region may be determined primarily

by the activity of genes in the region and/or its • Exonuclease activity can correct mistakes in DNA
state of compaction replication
▪ The less active, more tightly compacted the • Be able to describe important differences between
DNA, the later is the stage at which it is eukaryotic and prokaryotic DNA replication
replicated • Be able to name eukaryotic/prokaryotic replication
REPLICATION IN EUKARYOTES: THE REPLICATION enzymes and their function
FORK REPLICATION IN EUKARYOTES: MULTIPLE
• Events occurring at replication forks are very similar POLYMERASES
regardless of the genome; requires same collection of • Eukaryotes have five different DNA polymerases - α,
enzymes as prokaryotic fork β, γ, δ & ε;
o 3 are involved in nuclear replication; 2 are not
o A. All elongate DNA in a 5' —> 3' direction; none
of them is able tothe initiate synthesis of a DNA
chain without a primer
o B. Polymerases γ, δ & ε possess a 3' —> 5'
exonuclease.
• Polymerase γ - replicates mitochondrial DNA; not
involved in nuclear DNA replication
• Polymerase β - functions in DNA repair; not involved
in nuclear DNA replication
• Polymerase α - tightly bound to primase; together
they initiate the synthesis of each Okazaki fragment;
primase lays down a short primer; then polymerase α
extends it with ~20 deoxyribonucleotides
• Polymerase δ - assembles leading strand & most of
lagging strand fragments; thought to be the primary
replicative enzyme; requires "sliding clamp" structure
(PCNA), like polymerase III in E. coli
• Polymerase ε - determining its role has been difficult;
replication cannot be finished in cells lacking this
polymerase
REPLICATION IN EUKARYOTES: NUCLEAR
ORGANIZATION
• Replication apparatus consists of huge complex of
proteins operating within confines of a structured
nucleus;
• Increasing evidence suggests that replication
machinery is present in tight association with nuclear
matrix
o Replicating DNA moves like conveyer belt
through immobilized replication apparatus, rather
REVIEW FROM LAST TIME than remaining stationary
• Bacterial DNA replication: REPLICATION IN EUKARYOTES: CHROMATIN
o Know the functions of: STRUCTURE & REPLICATION
▪ DNA polymerases I, II, III • Nucleosomes form on both daughter duplexes very
▪ Helicase, primase, primosome near replication fork; indicates that assembly of DNA
▪ Β-clamps, γ-clamp loader, τ-subunits into nucleosomes is a very rapid event
▪ DNA ligase o 1. In vitro studies indicate that the (H3H4)2
• Be able to demonstrate and understanding how and tetramers present prior to replication remain
why the trombone model is used to describe DNA intact & are distributed randomly between 2
replication daughter duplexes
o 2. The 2 H2A/H2B dimers of each parental passed on and become a permanent part of the
nucleosome do not remain together as the population’s gene pool
replication fork moves through the chromatin o Somatic cells – not possible to pass the mutation
on but…
▪ 1. Can interfere with transcription and
replication
▪ 2. Can lead to the malignant transformation
of a cell
▪ 3. Can speed the process by which an
organism ages
• It is vital that cells have a way to repair the damage
and they do
o Damage is kept to <1 nucleotide/1000 bases
DNA REPAIR: NUCLEOTIDE EXCISION REPAIR
(NER)
• Removes part of strands having lesions:
o pyrimidine dimers & chemically altered
nucleotides;
o "cut-and-patch" mechanism;
o A. Transcription-coupled pathway - template
strands of genes that are being actively
transcribed are preferentially repaired; repair of
template strand is thought to occur as DNA is
being transcribed
▪ 1. The presence of the lesion may be
signaled by a stalled RNA polymerase
▪ 2. Ensures that those genes of greatest
importance to the cell, the genes being
actively transcribed, receive the highest
priority on the repair list
• B. Global pathway - slower, less efficient pathway
DNA REPAIR that corrects DNA strands in remainder of genome
• DNA is very susceptible to environmental damage • Steps in NER process in eukaryotic cells –
o 1. Lesion recognition by proteins scanning DNA
• A. Types of damage experienced by DNA
recognize distorted sites in helix
o 1. Ionizing radiation can break DNA backbone
o 2. The recruitment of repair enzymes to the lesion
o 2. Exposure to a variety of reactive chemicals can
o 3. The damaged strand is cut on both sides of the
alter DNA bases
lesion by a pair of endonucleases; the segment of
o 3. Ultraviolet radiation causes adjacent
damaged DNA is now held in position only by H
pyrimidines (C or T) to interact covalently
bonds
o 4. Thermal energy generated by metabolism in a
o 4. Segment of DNA between the incisions is
warm-blooded bird or mammal can split adenine
released
& guanine from their attachment to DNA
o 5. The gap is filled by DNA polymerase & the
backbone sugars
strand is sealed by DNA ligase
• B. Spontaneous alterations or lesions occur often
o Each cell of a warm-blooded mammal loses
~10,000 bases/day
DNA DAMAGE
• Potential effects
o Gametes – If damage occurs in a cell destined to
become a gamete, the damage (mutation) can be

o 3. Gap filled by DNA polymerase β
o 4. Strand is sealed by DNA ligase
• The cell recognizes alterations in the base. Thus, it

removes the base and not the entire nucleotide unlike
in the NER.
DNA REPAIR: MISMATCH REPAIR
• Cells replace mismatched bases that are
incorporated by DNA polymerase & escape the
enzyme's proofreading exonuclease
DNA REPAIR: BASE EXCISION REPAIR (BER)
• Mismatched base pairs cause distortions in double
• Sometimes single nucleotides in the double helix are helix geometry that are recognized by a repair
altered via chemical reaction and become enzyme
mismatched COMPLEX ISSUES IN DNA REPLICATION
• A. Uracil - forms by hydrolytic removal of cytosine's • 1. The helix must undergo localized unwinding, and
amino group the resulting “open” configuration must be stabilized
• B. 8-oxo-guanine -caused by damage from oxygen so that synthesis may proceed along both strands.
free radicals • 2. As unwinding and subsequent DNA synthesis
• C. 3-methyladenine -caused by alkylating agents; proceed, increased coiling creates tension further
transfer of methyl group from a methyl donor down the helix, which must be reduced.
• Steps in BER process in eukaryotes • 3. A primer of some sort must be synthesized so that
o Initiated by a DNA glycosylase that recognizes polymerization can commence under the direction of
alteration DNA polymerase III. Surprisingly, RNA, not DNA,
o DNA glycosylase removes the base (not the serves as the primer.
entire nucleotide) • 4. Once the RNA primers have been synthesized,
o The "beheaded" deoxyribose phosphate is DNA polymerase begins to synthesize the DNA
removed by (AP) endonuclease & DNA complement of both strands of the parent molecule.
polymerase Because the two strands are antiparallel to one
o 1. The AP endonuclease cleaves the DNA another, continuous synthesis in the direction that the
backbone replication fork moves is possible along only one of
o 2. Polymerase β removes the sugarphosphate the two strands. On the other strand, synthesis must
remnant that had been attached to the excised be discontinuous and thus involves a somewhat
base different process.
• 5. The RNA primers must be removed prior to
completion of replication. The gaps that are
temporarily created must be filled with DNA
complementary to the template at each location.
• 6. The newly synthesized DNA strand that fills each
temporary gap must be joined to the adjacent strand
of DNA.
• 7. While DNA polymerases accurately insert
complementary bases during replication, they are not
perfect, and, occasionally, incorrect nucleotides are
added to the growing strand. A proofreading
mechanism that also corrects errors is an integral
process during DNA synthesis.
COMPREHENSION CHECK
• 1. List all the proteins and enzymes involved in DNA
replication in prokaryotes and describe their
molecular functions.
• 2. Why is there a leading strand and a lagging strand?
• 3. Why is the synthesis of DNA runs from 5’ to 3’
indirection?
• 4. Why is there a need for an RNA primer in DNA
replication?
• 5. How does repair system know which member of a
mismatched pair is the incorrect nucleotide?
• 6. What are the similarities and differences of
eukaryotic and prokaryotic DNA replication?

WHAT BRIDGES DNA TO PROTEINS? In eukaryotic cell, the nucleus provides a separate
compartment for transcription. The original RNA
transcript called pre-mRNA is processed before
leaving the nucleus as mRNA.
mRNA bridges DNA to proteins
The overall process of formation of proteins will involve

the transcription of DNA to form RNA and the translation
of RNA to form protein. The process of gene expression
for eukaryotic and prokaryotic organisms may somewhat
differ. In prokaryotic organism, you don’t have any
nucleus or nuclear membrane that would separate the
genetic material from the cytoplasm. In the eukaryotic cell,
you have a nucleus that provides a separate compartment
for these processes. • Transcription
o is the synthesis of RNA using information in the
DNA
o “rewritten,” from DNA to RNA
o Forms messenger RNA (mRNA)
• Translation
o is the synthesis of a polypeptide using the
information in the mRNA
TRANSCRIPTION: RNA SYNTHESIS
INTRODUCTION
• The term gene has many definitions
• For this class, a gene is a segment of DNA used to
make a product that plays a functional role in the cell
In bacterial cell, mRNA produced from transcription in
o either an RNA or a polypeptide
immediately translated without additional
• Transcription is the first step in gene expression
processing
o It involves the reading of the DNA segment and
not the replication
WORDS TO KNOW
• Transcription: (Verb) The act or process of making a
copy
o Example: Court reporter hears the witness
speaking in English and types a written copy, in
English, of the witness’ statements.
▪ Transcriber
• Translation: Express the meaning of words or text
in another language
• Dogma: A principle or set of principles laid down
by an authority as incontrovertibly true
TRANSCRIPTION

• In genetics, the term refers to the copying of a DNA Transcription – is also known as RNA synthesis
sequence into an RNA sequence Replication – DNA synthesis
o Only one strand is copied, not the entire DNA You can actually compare and contrast replication from
• The structure of DNA is not altered as a result of this transcription and even replication from transcription from
process translation.
o It continues to store information and can be
transcribed again and again and again
GENE EXPRESSION
• Structural genes encode the amino acid sequence
of a polypeptide
o Transcription of a structural gene produces
messenger RNA, usually called mRNA
o The mRNA nucleotide sequence determines the
amino acid sequence of a polypeptide during
translation
o The synthesis of functional proteins
determines an organism’s traits
• This path from gene to trait is called the central
dogma of genetics
For it to be called gene expression, you have to take out

DNA replication. It involves transcription and translation.
OVERVIEW OF TRANSCRIPTION
• Gene expression is the overall process by which the
information within a gene is used to produce a
TRANSCRIPTION: THE DNA-DIRECTED SYNTHESIS
functional product which can, in concert with
OF RNA
environmental factors, determine a trait
• INITIATION
THE STAGES OF TRANSCRIPTION
• Transcription occurs in three stages
o Initiation
o Elongation
o Termination
• These steps involve protein-DNA interactions
o Proteins such as RNA polymerase interact with
DNA sequences
We have a lot of similarities of the processes of DNA
replication and RNA synthesis or DNA synthesis.

An RNA polymerase binds to a promoter. We have a • STEP 2
promoter region of a certain segment of DNA. In a gene,
we have this transcription unit and upstream of the
transcription unit is a promoter.
• ELONGATION
• STEP 3
• TERMINATION
TRANSCRIPTION ELONGATION
• RNA polymerase moves along the DNA template
strand, joining complementary RNA nucleotides to the
3’ end of the growing RNA transcript.
The DNA will recover or will from wounding again. • Behind the polymerase, the new RNA peels away
THE INITIATION OF TRANSCRIPTION AT A from the template strand, which re-forms a double
EUKARYOTIC PROMOTER helix with the non-template strand.
• STEP 1 The 3’ end is quite familiar because it is similar to the DNA
o In eukaryotic cells, proteins called transcription replication or DNA synthesis where nucleotides are joined
factors mediate the initiation of transcription by at the 3’ end and the synthesis runs from the 5’ to 3’
RNA polymerase II. directions.
The addition of nucleotide occurs at 3’ end.

TERMINATION OF TRANSCRIPTION • Spliceosomes
• The mechanism of termination differs between • Signal recognition particles
bacteria and eukaryotes. FUNCTIONS OF RNA MOLECULES
IN BACTERIA: Transciptomics – processes that occur during
• transcription proceeds through a terminator transcription process
sequence in the DNA
• transcribed terminator functions as the termination
signal, causing the polymerase to detach from the
DNA and release the transcript
• requires no further modification before
translation
IN EUKARYOTIC CELL:
• Pre-mRNA then undergoes further processing:
o 1. Alteration of mRNA ends
o 2. RNA splicing
RNA PROCESSING: ADDITION OF THE 5’ CAP AND
POLY-A TAIL.
addition of 5’ cap and Poly-A tail
Splicing involves the removal of introns leaving only

exons. Exons are the coding regions of the segment of a
gene while introns are non-coding regions of a gene.
Exons – are specific sites or specific DNA segments
where ribosomes will lead on.
RNA TRANSCRIPTS HAVE DIFFERENT FUNCTIONS
• Once they are made, RNA transcripts play different
functional roles
• Well over 90% of all genes are structural genes which
are transcribed into mRNA
o Final functional products are polypeptides
• The other RNA molecules in Table 12.1 are never
translated
o Final functional products are RNA molecules TRANSCRIPTION IN BACTERIA
• The RNA transcripts from nonstructural genes are not • Our molecular understanding of gene transcription
translated came from studies involving bacteria and
o They do have various important cellular functions bacteriophages
o They can still confer traits • Indeed, much of our knowledge comes from studies
o In some cases, the RNA transcript becomes part of a single bacterium
of a complex that contains protein subunits o E. coli
▪ For example • In this section we will examine the three steps of
• Ribosomes transcription as they occur in bacteria

PROMOTERS
• Promoters are DNA sequences that “promote” gene
expression
o More precisely, they direct the exact location
for the initiation of transcription
▪ It tells our RNA polymerase, “Hey! This is the
site of gene X codes for collagen..”
• Promoters are typically located just upstream of the
site where transcription of a gene actually begins
▪ That site is not actually transcribed. It is only
the binding site for RNA polymerase.
o The bases in a promoter sequence are
numbered in relation to the transcription start site
COMMON FEATURES OF PROMOTERS IN E. COLI
Quotation Box (upper left): Most of the promoter region
• There are two consensus sequences at −10
is labelled with negative number
(TATAAT) and −35 (TTGACA) for σ subunit binding.
Quotation Box (upper right): Bases preceding the start
o called TATA sequences
site are numbered direction. There is no base numbered
▪ -10 means 10 nucleotides away or 10
0
nucleotides before the start of the
Quotation Box (lower): Bases to the right are numbered
transcription site
in a positive direction
▪ σ is a portion of the RNA polymerase that
specifically binds to the promoter that helps
direct RNA polymerase to bind to that side.
• An A-T−rich upstream promoter element between
−40 and −60 binds the α subunit.
• A-T−rich sequences promote strand separation.
• These sequences govern efficacy of RNA Pol binding
and therefore affect gene-expression level.
• Nucleotides before the first nucleotide of the RNA
molecule are considered “upstream” and given
negative values.
EXAMPLES OF E. COLI PROMOTER REGIONS
Quotation Box (upper left): Sequence elements that

play a key role in transcription
Quotation Box (upper right): The promoter may span a
large region, but specific short sequence elements are
particularly critical for promoter recognition and activity
level
Purple regions = promoters Quotation Box (lower): Sometimes termed the Pribnow
box, after its discoverer
-10 sequence is where the segment or a subunit
of RNA polymerase will bind to help initiate transcription.
INITIATION OF BACTERIAL TRANSCRIPTION
• RNA polymerase is the enzyme that catalyzes the
synthesis of RNA
• In E. coli, the RNA polymerase holoenzyme is
composed of
o Core enzyme

▪ Five subunits = α2ββ’ω ▪ A region within the sigma factor that
o Sigma factor contains a helix-turn-helix structure is
▪ One subunit = σ involved in a tighter binding to the DNA
o These subunits play distinct functional roles BINDING OF σ FACTOR PROTEIN TO DNA DOUBLE
RNA POLYMERASE IS A LARGE ENZYME WITH NO HELIX
PROOFREADING CAPABILITY
• RNA polymerase holoenzyme has five core subunits
of α2ββω plus a sixth called σ.
o σ binds to the promoter
• See Figure 26-4.
• RNA Pol lacks 3’ → 5’ - exonuclease, so it has a
high error rate of 1/104–1/105.
• RNA binds to promoter regions to initiate
transcription.
• The binding of the RNA polymerase to the promoter

forms the closed complex
• Then, the open complex is formed when the
TATAAT box in the -10 region is unwound
• A short RNA strand is made within the open complex
o The sigma factor is released at this point
o This marks the end of initiation
• The core enzyme now slides down the DNA to
synthesize an RNA strand
o This is known as the elongation phase
BACTERIAL RNA POLYMERASE HAS AT LEAST SIX

SUBUNITS
• Two α subunits function in assembly and binding to
UP (upstream promoter) elements.
o See slide on promoters and Fig. 26-5
• The β subunit is the main catalytic subunit.
o Carries out the actual process of making the RNA
or polymerizing RNA.
• The β’ subunit is responsible for DNA binding.
• The σ subunit directs enzyme to the promoter.
o Each class of RNA pol holoenzymes has a
different s subunit.
• The ω (omega) appears to protect the polymerase
from denaturation.
INITIATION OF BACTERIAL TRANSCRIPTION
• The RNA polymerase holoenzyme binds loosely to
the DNA
• It then scans along the DNA, until it encounters a
promoter region
o When it does, the sigma factor recognizes both
the – 35 and –10 regions

ELONGATION IN BACTERIAL TRANSCRIPTION • The open complex formed by the action of RNA
• The RNA transcript is synthesized during the polymerase is about 17 bases long
elongation stage o Behind the open complex, the DNA rewinds back
• The DNA strand used as a template for RNA into a double helix
synthesis is termed the template strand • On average, the rate of RNA synthesis is about 43
• The opposite DNA strand is called the coding strand nucleotides per second!
o It has the same base sequence as the RNA
transcript
▪ Except that T in DNA corresponds to U in
RNA
• In transcription, RNA polymerase reads only one
strand of the DNA
o It reads the template strand
o It moves along the template strand 3’ to 5’
• The RNA polymerase simultaneously makes a RNA
copy of the template strand’s complementary partner
o The partner strand is called the coding strand
o The new mRNA molecule is made in the 5’ to 3’
direction
o The orientation and sequence of the mRNA is
identical to the coding strand (except U’s for T’s)
TEMPLATE VERSUS CODING STRAND
• DNA template strand – serves as template for RNA
polymerase
• DNA coding strand –the non-template strand; has
the same sequence as the RNA transcript
o When we are referring to the coding strand, we KEY POINTS
are referring to the RNA not the RNA. • RNA polymerase slides along the DNA, creating an
***Regulatory sequences are listed by the coding strand open complex as it moves.
sequence. • The DNA strand known as the template strand is
used to make a complementary copy of RNA as an
RNA–DNA hybrid.
• RNA polymerase moves along the template strand in
a 3′ to 5′ direction, and RNA is synthesized in a 5′ to
3′ direction using nucleoside triphosphates as
precursors. Pyrophosphate is released (not shown).
(SIMILAR TO THE SYNTHESIS OF DNA VIA DNA
POLYMERASE)
• The complementarity rule is the same as the AT/GC
rule except that U is substituted for T in the RNA.
TERMINATION OF BACTERIAL TRANSCRIPTION
• Termination is the end of RNA synthesis
o It occurs when the short RNA-DNA hybrid of the
open complex is forced to separate
▪ This releases the newly made RNA as well as
the RNA polymerase
• E. coli has two different mechanisms for termination
o 1. rho-dependent termination
▪ Requires a protein known as r (rho)
o 2. rho-independent termination
▪ Does not require r
TWO TYPES OF TERMINATION IN E. COLI
• ρ-independent – characterized by three U’s near the
3’ end of the transcript
o self-complementary regions in transcript form a
hairpin 15−20 nt before the 3’ end
▪ makes the RNA Pol pause, dissociate
3U’s are read, forming a hairpin loop that causes

destabilization of the complex. mRNA is released and
polymerase is dissociated.
• ρ-dependent
o common CA-rich sequence called a rut site (Rho
utilization element)
o ρ protein processes until termination site reached
o ρ protein is a helicase, binds to rut site
Rho utilization element is transcribed, it attracts the rho

protein. Rho helicase moves towards the 3’ end of the
transcript. The RNA polymerase reaches the terminator.
A stem-loop causes RNA polymerase to pause. RNA
polymerase pauses due to its interaction with the stem-
loop structure. Rho protein catches up to the open
complex and separates the RNA-DNA hybrid. RNA-DNA
hybrid forms a double helix or wounding or binding • For structural genes, at least three features are found
together. The helicase will unwound or separate the in most promoters
hybrid to release mRNA. o Regulatory elements
• ρ-independent termination is facilitated by two o TATA box
sequences in the RNA o Transcriptional start site
o 1. A uracil-rich sequence located at the 3’ end of
the RNA
o 2. A stem-loop structure upstream of the uracil-
rich sequence
• The core promoter is relatively short

o It consists of the TATA box and transcriptional
start site
▪ Important in determining the precise start
point for transcription
• The core promoter by itself produces a low level of
transcription
o This is termed basal transcription
TRANSCRIPTION IN EUKARYOTES
• Many of the basic features of gene transcription are
very similar in bacteria and eukaryotes • Regulatory elements are short DNA sequences that
• However, gene transcription in eukaryotes is more affect the binding of RNA polymerase to the
complex promoter
o Larger, more complex cells (organelles) o are usually upstream from the transcription start
o Added cellular complexity means more genes site.
that encode proteins are required • Transcription factors (proteins) bind to these
o Multicellularity adds another level of regulation elements (regulatory elements) and influence the rate
▪ express genes only in the correct cells at the of transcription
proper time Not all genes are transcribed in a certain cell. It depends
EUKARYOTIC RNA POLYMERASES on the transcription factor found within that cell.
• Nuclear DNA is transcribed by three different RNA o There are two types of regulatory elements
polymerases ▪ Enhancers
o RNA pol I • Stimulate transcription
▪ Transcribes all rRNA genes (except for the ▪ Silencers
5S rRNA) • Inhibit transcription
• RNA pol II o They vary widely in their locations but are often
o Transcribes all structural genes found in the –50 to –100 region
▪ Thus, synthesizes all mRNAs • Factors that control gene expression can be divided
o Transcribes some snRNA genes into two types, based on their “location”
• RNA pol III • cis-acting elements
o Transcribes all tRNA gene o DNA sequences that exert their effect only over
o And the 5S rRNA gene a particular gene
SEQUENCES OF EUKARYOTIC STRUCTURAL o Example: TATA box, enhancers and silencers
GENES • trans-acting elements
• Eukaryotic promoter sequences are more variable o Regulatory proteins that bind to such DNA
and often more complex than those of bacteria sequences

RNA POLYMERASE II AND ITS TRANSCRIPTION o The sequence of codons in the mRNA provides
FACTORS the instructions for the sequence of amino acids
• Three categories of proteins are required for basal in the polypeptide
transcription to occur at the promoter • This is termed the colinearity of gene expression
o RNA polymerase II o Because the sequence of DNA is actually the
o Five different proteins called general same as that of the mRNA produced
transcription factors (GTFs) • Analysis of eukaryotic structural genes in the late
o A protein complex called mediator (we won’t go 1970s revealed that they are not always colinear
over this) with their functional mRNAs
• Figure 12.14 shows the assembly of transcription o Proves that mRNAs produced after transcription
factors and RNA polymerase II at the TATA box are modified
• Instead, coding sequences, called exons, are
interrupted by intervening sequences or introns
o Exons are coding sequence read by the
ribosomes to form polypeptide sequence. The
sequences between the exons (introns) are not
read by the ribosomes so they have to be
removed.
• Transcription produces the entire gene product
o Introns are later removed or excised
o Exons are connected together or spliced
• This phenomenon is termed RNA splicing
o It is a common genetic phenomenon in
eukaryotes
o Occurs occasionally in bacteria as well
• Aside from splicing, RNA transcripts can be modified
in several ways
Point: we utilize different transcription factors to the o For example
promoter side. ▪ Trimming of rRNA and tRNA transcripts
RNA POL II TRANSCRIPTIONAL TERMINATION ▪ 5’ Capping and 3’polyAtailing of mRNA
• Pre-mRNAs are modified by cleavage near their 3’ transcripts
end with subsequent attachment of a string of
adenines
• Transcription terminates 500 to 2000 nucleotides
downstream from the poly A signal
• There are two models for termination
o Further research is needed to determine if either,
or both are correct (we won’t cover this)
RNA MODIFICATION
• Post-transcriptional modification
• All of the RNAs produced in eukaryotes undergo
some series of RNA modification. It will not
immediately led by the ribosome.
• Analysis of bacterial genes in the 1960s and 1970
revealed the following:
o The sequence of DNA in the coding strand
corresponds to the sequence of nucleotides in
the mRNA

SPLICING Get rid of the introns before it is read by the ribosome.
• Three different splicing mechanisms have been Then, it leaves the nucleus.
identified PRE-mRNA SPLICING
o Group I intron splicing • The spliceosome is a large complex that splices pre-
o Group II intron splicing mRNA
o Spliceosome (we’ll focus on this mechanism) • It is composed of several subunits known as snRNPs
• All three cases involve (pronounced “snurps”)
o Removal of the intron RNA o Each snRNP contains small nuclear RNA and a
o Linkage of the exon RNA by a phosphodiester set of proteins
bond • The subunits of a spliceosome carry out several
functions
o 1. Bind to an intron sequence and precisely
recognize the intron-exon boundaries
o 2. Hold the pre-mRNA in the correct
configuration
o 3. Catalyze the chemical reactions that remove
introns and covalently link exons
• Intron RNA is defined by particular sequences within
the intron and at the intron-exon boundaries
• The consensus sequences for the splicing of
mammalian pre-mRNA are shown in the following:
• In eukaryotes, the transcription of structural genes

produces a long transcript known as pre-mRNA
o Pre-mRNA is the product once the RNA
polymerase II of eukaryotes reads the DNA
template from 3’ to 5’
• This RNA is altered by splicing and other
modifications, before it leaves the nucleus
• Splicing in this case requires the aid of a
multicomponent structure known as the spliceosome
U1 binds with 5’. U2 binds to the branching site.

▪ It is estimated that about 70% are
alternatively spliced
▪ Note: Certain mRNAs can be alternatively
spliced to produce dozens of different
mRNAs
• The next image considers an example of alternative
splicing for a gene that encodes α-tropomyosin
o This protein functions in the regulation of cell
contraction
o It is found in
▪ Smooth muscle cells (uterus and small
intestine)
▪ Striated muscle cells (cardiac and skeletal
muscle)
▪ Also in many types of non-muscle cells at low
INTRON ADVANTAGE? levels
• One benefit of genes with introns is a phenomenon • The different cells of a multicellular organism regulate
called alternative splicing contractibility in subtly different ways
• A pre-mRNA with multiple introns can be spliced in o One way to accomplish this is to produce different
different ways forms of α-tropomyosin by alternative splicing
o This will generate mature mRNAs with different
combinations of exons
• This variation in splicing can occur in different cell
types or during different stages of development
• The biological advantage of alternative splicing is that
two (or more) polypeptides can be derived from a
single gene
• This allows an organism to carry fewer genes in its
genome
ALTERNATIVE SPLICING
• One very important biological advantage of introns in
eukaryotes is the phenomenon of alternative splicing
• Alternative splicing refers to the phenomenon that
pre-mRNA can be spliced in more than one way
o Alternatively splicing produces two or more The main point of alternative splicing is to create diversity
polypeptides with different amino acid sequences of the transcript.
o In most cases, large sections of the coding CAPPING
regions are the same, resulting in alternative • Most mature mRNAs have a 7-methylguanosine
versions of a protein that have similar functions covalently attached at their 5’ end
o Nevertheless, there will be enough differences in o This event is known as capping
amino acid sequences to provide each • Capping occurs as the pre-mRNA is being
polypeptide with its own unique characteristics synthesized by RNA pol II
• The degree of splicing and alternative splicing varies o Usually when the transcript is only 20 to 25 bases
greatly among different species long
• Baker’s yeast contains about 6,300 genes • As shown in Figure 12.23, capping is a three-step
o ~ 300 (i.e., 5%) encode mRNAs that are spliced process
▪ Only a few of these 300 have been shown to
be alternatively spliced
• Humans contain ~ 25,000 genes
o Most of these encode mRNAs that are spliced

GTP to form GMP releases phosphate group called
pyrophosphate and the resulting GMP will bind to the
phosphate group.
• The 7-methylguanosine cap structure is
recognized by cap-binding proteins
• Cap-binding proteins play roles in the
o Movement of some RNAs into the cytoplasm
o Early stages of translation
o Splicing of introns
TAILING
• Most mature mRNAs have a string of adenine
nucleotides at their 3’ ends
o This is termed the polyA tail
• The polyA tail is not encoded in the gene sequence
o It is added enzymatically after the gene is
completely transcribed
• The attachment of the polyA tail is shown in Figure
12.24
We have now the transcript (upper left). At the 5’ end,

there is a phosphate. The phosphate group will be the
substrate for the RNA 5’-triphosphatase which removes
an extra phosphate from the 5’ end.
There is a consensus sequence in higher eukaryotes

once an mRNA is formed. The endonuclease in the
nucleus cleavage occurs about 10 nucleotides
downstream from the sequence (indicated on the first red
strand). Endonuclease cuts (3’ portion) a portion when it
recognizes polyadenylation signal sequence. Then, it
adds a lot of adenine nucleotides at 3’ portion.
TRANSLATION: PROTEIN SYNTHESIS
Translation: RNA-directed synthesis of a polypeptide
• As a molecule of mRNA is moved through a
ribosome, codons are translated into amino acids,
one by one.
• The interpreters are tRNA molecules, each type
with a specific nucleotide triplet called an anticodon
at one end and a corresponding amino acid at the
other end.

• A tRNA adds its amino acid cargo to a growing
polypeptide chain when the anticodon hydrogen
bonds to the complementary codon on the mRNA.
Key Role Players in Translation Process: mRNA with

tRNA that is bounded to an amino acid (its product is a
polypeptide)
FEATURES OF PROTEIN SYNTHESIS
THE ELONGATION CYCLE OF TRANSLATION.
• Large energy cost
• Can be rapid when accomplished on clusters of
ribosomes called a polysome
• In bacteria, tightly coupled to transcription
o Translation can begin before transcription is
finished.
▪ Bacteria do not have any compartments.
Ribosomes are readily available to immediate
read the mRNA that is produced from
transcription.

It has two subunits. The larger subunit consists of sites:
A, P, E. It is on the P-site, there is an exit tunnel. It is
where proteins will be released from.
THE TERMINATION OF TRANSLATION.

THE GENETIC CODE IS UNIVERSAL, WITH A FEW
EXCEPTIONS
• It is used by prokaryotes and eukaryotes, across
species.
• Mitochondria contain DNA and use a slightly different
code.
o UGA encodes Trp in vertebrate mtDNA (instead
of STOP).
o AGA/AGG encodes STOP in vertebrate mtDNA
(instead of Arg).
• Mitochondria encode their own tRNAs, using 22
instead of 32.
“WOBBLE” PAIRINGS IN TRNA WITH mRNA CAN
OCCUR IN THE THIRD BASE
CODONS: TRIPLET OF BASES
• The third base of a codon can form noncanonical
• The flow of information from gene to protein is
base pairs with its complement (anticodon) in tRNA.
based on a triplet code: a series of nonoverlapping,
• Some tRNAs contain Inosinate (I), which can H-bond
three-nucleotide words
with U, C, and A.
• These triplets are the smallest units of uniform length
o These H-bonds are weaker and were named by
that can code for all the amino acids
Crick as “wobble” base pairs.
• During transcription, one of the two DNA strands
o Example: In yeast, CGA, GCU, and CGC all bind
called the template strand provides a template for
to tRNAArg, which has the anticodon 3’-GCI-5’.
ordering the sequence of nucleotides in an RNA
▪ Although sequences are usually written
transcript
5’→3’, the anticodon here is written 3’→5’to
• During translation, the mRNA base triplets, called
illustrate its bonding to the mRNA codons.
codons, are read in the 5’ to 3’ direction
MOLECULAR RECOGNITION OF CODONS IN mRNA
• Each codon specifies the addition of one of 20 BY tRNA
amino acids
• The codon sequence is complementary with the
anticodon sequence.
• The codon in mRNA base pairs with the anticodon in
mRNA via hydrogen bonding.
• The alignment of two RNA segments is antiparallel.
The third nuclear base has the capacity to be changed,

to be varied.

THE GENETIC CODE IS RESISTANT TO MUTATIONS

FIVE STAGES OF PROTEIN SYNTHESIS
• Degenerate code allows certain mutations to still code
• 1. Activation of amino acids
for the same amino acid.
o tRNA is aminoacylated
o “silent” mutations―different nucleotide in DNA
but same amino acid in protein • 2. Initiation of translation
o The mRNA and aminoacylated tRNA bind to
• Mutation in the first base of a codon usually produces
ribosome bind to the small ribosomal subunit. The
a conservative substitution.
large subunit then binds.
o Example: GUU→Val but AUU→Leu
SOME mRNAS ARE EDITED BEFORE PROTEIN • 3. Elongation
SYNTHESIS o Successive cycles of aminoacyl-tRNA binding
and peptide bond formation…until the ribosome
• Editing involves alteration, addition, or deletion of
reaches a STOP codon
nucleotides in mRNA.
o Before mRNAs are led by ribosomes • 4. Termination and ribosome recycling
o Translation stops when a stop codon is
• Editing uses guide RNAs (gRNAs) that temporarily
encountered. The mRNA and protein dissociate,
hybridize with the mRNA and act as templates for
and the ribosomal subunits are recycled
editing.
• 5. Folding and posttranslational processing
o catalyzed by a variety of enzymes
o Protein folding and posttranslational processing
OVERVIEW OF PROTEIN SYNTHESIS
DEAMINATION REACTIONS YIELD

• RNA editing by alteration of nucleotides most
commonly involves the enzymatic deamination of
adenosine or cytidine residues.

Components Required for the Five Major Stages THE RIBOSOME IS A KEY PLAYER IN PROTEIN
of Protein Synthesis in E. Coli SYNTHESIS
STAGE ESSENTIAL • Ribosomal RNA is the major composition of ribosome
COMPONENTS and not protein.
Activation of amino 20 amino acids • Make up ~25% of dry weight of bacteria
acids 20 aminoacyl-tRNA • ~65% rRNA, 35% protein
syntheses o rRNA forms the core.
32 or more tRNAs o RNA does the catalysis of peptide bond
ATP formation.
Mg2+ • Made of two subunits bound together (30S and 50S)
Initiation mRNA in bacteria, with mRNA running through them
N-Formylmethionyl-
tRNAfMet
Initiation codon in mRNA
(AUG)
30S ribosomal subunit STRUCTURE OF RIBOSOMES IN BACTERIA AND
50S ribosomal subunit YEAST
Initiation factors (IF1,
IF2, IF3)
GTP
Mg2+
Elongation Functional 70S
ribosomes (initiation
complex)
Aminoacyl-tRNAs
specified by codons
Left: ribosome structure in bacteria
Elongation factors (EF-
Right: ribosome structure in yeast
Tu, EF-Ts, EF-G)
rRNAs HAVE COMPLEX SECONDARY STRUCTURES
GTP
Mg2+
Termination and Termination codon in
ribosome recycling mRNA
Release factors (RF1,
RF2, RF3, RRF)
EF-G
IF3
Folding and Chaperones and folding
posttranslational enzymes (PPI, PDI);
processing specific enzymes,
cofactors, and other
components for removal
of initiating residues and
signal sequences,
additional proteolytic
processing, modification
of terminal residues, and
attachment of acetyl, • The three ss rRNAs of E.coli have specific 3-D
phosporyl, methyl, structure with extensive intrachain base pairs.
carboxyl, carbohydrate, • Shapes of rRNAs are highly conserved.
or prosthetic groups o They are not easily changed from one species to
another.
RIBOSOMES IN BACTERIA AND EUKARYOTES
• Overall, very similar
• Still two subunits with mRNA running between
• In eukaryotes, larger (80S), more complex, contain >
80 proteins
• Chloroplasts and mitochondria have ribosomes
simpler than those in bacteria.
3-D STRUCTURE OF YEAST tRNAPhe
CHARACTERISTICS OF tRNAs
Left: three-dimensional structure of the yeast, tRNA
• ssRNA of 73–93 nucleotides in both bacteria and
containing phenylalanine
eukaryotes
RECALL FIVE STAGES OF PROTEIN SYNTHESIS
• Cloverleaf structure in 2-D
• 1. Activation of amino acids
• “Twisted L” shape in 3-D o tRNA aminoacylated
• Most have G at 5’-end; all have CAA at 3’-end • 2. Initiation of translation
• Have modified bases o mRNA and aminoacylated tRNA bind to ribosome
o methylated bases, and so on • 3. Elongation
• Amino acid arm o cycles of aminoacyl-tRNA binding and peptide
o has amino acid esterified via carboxyl group to bond formation…until a STOP codon is reached
the 2’-OH or 3’-OH of the A of the terminal CAA • 4. Termination and ribosome recycling
codon o mRNA and protein dissociate, ribosome recycled
• Anticodonarm • 5. Folding and posttranslational processing
• D arm o catalyzed by a variety of enzymes
o contains dihydrouridine (D) STAGE 1 –ACTIVATION OF AMINO ACIDS
o contributes to folding • Step 1 − creation of aminoacyl intermediate
• TᴪC arm o Aminoacyl-tRNA synthetases esterify 20 amino
o contains pseudouridine (ᴪ)―has bonding acids to corresponding tRNAs.
between base and ribose
o helps in folding

▪ COO– of amino acid attacks phosphate of • Some cells contain fewer than 20 synthetases; in this
ATP→creates aminoacyladenylate case, one amino acid is converted to another after
intermediate charging the tRNA.
o Pyrophosphate (PPi) is also cleaved, so the
reaction is driven forward by two
phosphoanhydride bond cleavages.
o The fate of the aminoacyladenylate varies.
AMINOACYLATION OF tRNA
THE SECOND GENETIC CODE

• Step 2 – transfer of aminoacyl to tRNA • Aminoacyl-tRNA synthetases must be specific for
o Aminoacyl-tRNA synthetases (two classes) both amino acid and tRNA.
transfer aminoacyl group from enzyme to tRNA. o Matching each amino acid with correct tRNA can
▪ 2’-OH or 3’-OH of tRNA attacks phosphate of be viewed as the “second genetic code.”
aminoacyl intermediate, creating o The “code” is in molecular recognition of a
phosphodiester bond between amino acid specific tRNA molecule by a specific synthetase.
and tRNA • Only a few nucleotides in tRNA confer the binding
AMINOACYLATION OF tRNA specificity.
o anticodon region
o The primary determinant in Ala-tRNA is a single
G=U in the amino acid arm.
NUCLEOTIDE POSITIONS IN TRNAS THAT ARE
RECOGNIZED BY AMINOACYL-tRNA
SYNTHETASES: (ORANGE AND GREEN POINTS)
AMINOACYL-tRNA SYNTHETASES
• Each enzyme binds a specific amino acid and the
matching tRNA.
• Most cells contain 20 different aminoacyl-tRNA STAGE 2: INITIATION (PROKARYOTES)
synthetases, one for each amino acid.
• The first tRNA is unique. • In bacteria, initiation requires:
• The first codon of any peptide is AUG (Met). o 30S ribosomal subunit
• All organisms have two tRNAs for Met. o mRNA
o Chloroplasts are found in plants. Mitochondria is o fMet-tRNA
found in eukaryotes. o initiation factors IF-1, IF-2, and IF-3
o In bacteria, plus chloroplasts and o GTP
mitochondria, initiation tRNA inserts N- o 50S ribosomal subunit
formylmethionine (uses a special tRNA ). fMet o GTP
o Interior Met is inserted with normal tRNAMet. o Mg2+
• Eukaryotic protein begins with Met, not fMet, but a • Step 1: The 30S ribosomal subunit binds IF-1, IF-
special tRNA is still used for peptide initiation. 2, and IF-3 and mRNA.
o Methionine is actually unique because it is the o Initiation factor IF-3 keeps 30S and 50S subunits
first codon. It is the first amino acid that is placed apart.
in any protein. o The initiating (5’)-AUG codon of mRNA is guided
UNUSUAL AMINO ACIDS FOUND IN PEPTIDES to its correct position by the Shine−Dalgarno
• Twenty genetically encoded amino acids are sequence (region in mRNA that is
common in all organisms. complementary to a sequence in ribosomal
• But two additional amino acids are also found in a few RNA).
proteins and are genetically coded. • Step 2: fMet-tRNAfMet joins the complex.
• Selenocysteine o Formylmethionine tRNA binds to the peptidyl (P)
o formed after charging an UGA(stop)-recognizing site along with initiating (5’) AUG.
tRNA with serine in both bacteria and eukaryotes
o this unique tRNA found at low levels in the cell
• Pyrrolysine
o directly attached to its tRNA that recognizes
UAG(stop) codon by some archae
• Step 3: 50S subunit associates

o Large 50S subunit combines with the 30S subunit
forming the initiation complex.
▪ IF-2 hydrolyzes GTP.
STAGE 2: INITIATION (PROKARYOTIC)

• Initiation requires a large assembly.

• Step 3: mRNA binds with eIF4F

o eIF4F includes
o eIF4E (binds the 5’cap)
o eIF4A (an ATPase and RNA helicase)
mRNA SEQUENCES THAT SERVE AS SIGNALS FOR o eIF4G (linker protein which binds to PABP –
INITIATION OF PROTEIN SYNTHESIS IN BACTERIA poly(A) binding protein at the 3’ poly (A) tail)
Shine-Dalgarno sequence are signals for initiation of

protein synthesis.
STAGE 2: INITIATION (EUKARYOTES)
• Use more initiation factors
o over 12, including eIFIA and eIF3 (functional
homologs of IF-1 and IF-3)
• Has different mechanistic details
• Has a step that circularizes the mRNA during initiation
• Step 1: 40S subunit joins with eIF1A and eIF3
o eIF1 binds to the E site, eIF1A binds to the A site,
EIF3 blocks the A site preventing tRNA binding
• Step 2: eIF2 and the charged tRNAMet with bound • Step 4: scanning of the mRNA until an AUG codon
GTP join the 40S is found
o creating the 43S preinitiation complex • Step 5: 60S subunit associates and many of the
initiation factors are released.
The first peptide bond starts with fMet and the incoming
second amino acid will bind to the p site. The dehydration
reaction also occurs in the peptide bond formation.
• Step 3: translocation of the ribosome
o The ribosome moves one codon toward the 3’-
end of the mRNA.
▪ uses energy from GTP hydrolysis
• GTP is part of EF-G (translocase)
▪ leaves A site open for new aminoacyl-tRNA
STAGE 3: ELONGATION (PROKARYOTIC)

• Step 1: binding of the incoming aminoacyl-tRNA
o Incoming aminoacyl-tRNA binds first to an EF-Tu
–GTP complex.
o The aminoacyl-EF-Tu-GTP complex binds to the
aminoacyl (A) site of the 70S initiation complex.
o After GTP hydrolysis, EF-Tu-GDP leaves the
ribosome.
• Step 2: peptide bond forms
o There are now two amino acids bound to tRNAs
positioned for joining.
▪ One is on the A site, the other on the P site.
o N-formylmethionyl group is transferred from its
tRNA in the P site to the amino acid in the A site.
▪ The reaction is catalyzed by the 23S rRNA
(ribozyme).
o “Uncharged” (deacetylated) tRNAfMet is now in the
P site. The ribosome is moving and not the mRNA.
FORMATION OF THE FIRST PEPTIDE BOND STAGE 3: ELONGATION (EUKARYOTIC)
• Steps are similar to bacteria
• Elongation factors –eEF1α(EF-Tu), eEF1βγ(EF-Ts),
eEF2 (EF-G)
• Difference: Eukaryotic ribosomes do NOT have an E
site; the uncharged tRNAs are released from the P
site.
STAGE 4: TERMINATION
• Signaled by a stop codon

• UAA, UAG, or UGA in the A site will trigger the action o Translation continues until a stop codon is found
of termination factors (release factors) RF-1, RF-2, in the tmRNA.
RF-3. o The defective mRNA and polypeptide are both
• This helps to: degraded.
o hydrolyze terminal peptide-tRNA bond
o release peptide and tRNA from ribosome
o cause subunits of ribosome to dissociate so that
initiation can begin again
STAGE 5: POSTTRANSLATIONAL MODIFICATIONS

• Some proteins require modification before the fully
active conformation is achieved.
• Posttranslational modifications include:
RIBOSOME RESCUE
o enzymatic removal of formyl group from first
• Damaged mRNA lead to the formation of an
residue, or removal of Met and sometimes
incomplete or peptide and a “nonstop complex.”
additional residues
• The ribosome is rescued by trans-translation. o acetylation of N-terminal residue
o A transfer-messenger RNA (tmRNA) and small
• Removal of signal sequences or other regions
protein B (SmpB) bind to the stalled complex in
• Attaching carbohydrates
the empty A site.

• Removing sequence to activate an enzyme
POSTTRANSLATIONAL MODIFICATIONS
• Modifying amino acids with additional phosphates,
carboxylic acid groups, and so on
• Addition of isoprenyl groups (such as farnesyl
pyrophosphate) from intermediates of cholesterol
synthesis pathway
o Isoprene or derived group helps anchor proteins
in membranes.
• Adding prosthetic groups
• Forming disulfide links
MORE MODIFIED AMINO ACIDS

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BIOCHEMISTRY FIRST SEMESTER

LECTURE | CHRISTIAN JOHN CAPIRIG, MD | MIDTERM A.Y. 2021 - 2022

TELETUBBIES AND FRIENDS 1

• Stereoisomers differ from each other in configuration.

TELETUBBIES AND FRIENDS 2

This is what happens as another carbon is added to

TELETUBBIES AND FRIENDS 3

TELETUBBIES AND FRIENDS 4

A comparison of the Fischer, complete Haworth, and

TELETUBBIES AND FRIENDS 5

Structures of two deoxy sugars. The structures of the

Two well-known alditols are economically important:

The hydroxyl group or any hydroxyl

TELETUBBIES AND FRIENDS 7

Two different disaccharides of α-D-glucose. These

TELETUBBIES AND FRIENDS 8

Reducing sugars. A disaccharide with a free hemiacetal

TELETUBBIES AND FRIENDS 9

• If you try to test for reducing sugars for sucrose (the

TELETUBBIES AND FRIENDS 1

• Build-up of lactose causes bacterial lactase to act on

TELETUBBIES AND FRIENDS 2

TELETUBBIES AND FRIENDS 3

The structure of starch is based on the α-anomer of

TELETUBBIES AND FRIENDS 4

A core protein of glycogenin is surrounded by branches

• Chitin: polysaccharide similar to cellulose in both

TELETUBBIES AND FRIENDS 5

The structure of the peptidoglycan of the bacterial cell

Glycosaminoglycans, which are formed from repeating

*Each polymer is classified as a homopolysaccharide

TELETUBBIES AND FRIENDS 7

Carbohydrates present in the plasma membrane as short,

TELETUBBIES AND FRIENDS 8

CARBOHYDRATES AND THE IMMUNE RESPONSE

TELETUBBIES AND FRIENDS 9

TYPES OF LIPIDS: FATTY ACIDS

TELETUBBIES AND FRIENDS 1

• Based on number of C=C double bonds,

• The image below shows us that fatty acids are

FATTY ACIDS – ESTERIFIED WITH THE GLYCEROL

TELETUBBIES AND FRIENDS 2

TYPES OF LIPIDS: TRIGLYCERIDES

• If the fatty acid is in Cis form, it will exhibit “kinking”

• Triglycerides that contain saturated fatty acids pack

• Is the process to convert a liquid oil (with kinks in its

TELETUBBIES AND FRIENDS 3

TYPES OF LIPIDS: PHOSPHOLIPIDS

• Take a look at the glycerol in the left side. OH is basic.

• Hydrophobic – no polar charges or hydrophilic groups

TELETUBBIES AND FRIENDS 4

• Composed of 4 fused hydrophobic rings (A,B,C

NAME THAT LIPID

• Look at the image above. We will discuss theme one

TELETUBBIES AND FRIENDS 6

• Proteins are hydrophilic in nature, but why can they

TELETUBBIES AND FRIENDS 7

• Phospholipid is acted upon by the phospholipase

• Take a look at the picture above.

• Take a look at the CM (chylomicron) that is formed.

TELETUBBIES AND FRIENDS 9

• Refer to the picture above for a quick recap of the