LESSON 1: Gregor Mendel

INTRODUCTION TO - Characters are controlled by factors

MOLECULAR BIOLOGY - Discovery of the inheritance of biological
traits
Wilhelm Johannsen
Molecular biology - branch of biological
science that explain processes in terms of - Coined the term gene 1909
molecular interaction.
Thomas Morgan
- Study of biology and molecular level
- observe the gene associated with
- Biochemistry of genes and their
chromosomes in 1910
products.
Frederick Griffith 1928
BASIC STREAMS OF SCIENCE
- Transformation in diplococcus
- Genetics - deals with identification and
pneumonia
characterization of genes and also the
- DNA was the transmittable genetic
inheritance of genes
material.
- Cell biology - concerns with this doctors
and functions of the cell organelles M. Schlesinger 1934
- Biochemistry - involves the pointing out
- Bacteriophages are compose of DNA and
of 3 dimensions structures and actions of
protein
different macromolecules.
 Molecular biology was coined by George Beadle, Edward Tatum 1941
Warren Weaver 1938 (a director of
natural science division of - Published the results of biochemical
Rockefeller Foundation) -- describe genetics of neurospora
the program, application of tools, - Established one gene-one enzyme
physical science to biology, hypothesis
biochemistry, and genetics. Oswald Avery, Colin MacCleod, Maclyn
McCarty 1944
 First use by William Astbury 1945 to
study chemical physical structure of - Transforming principle of streptococcus
by logical macromolecules pneumoniae was DNA

Erwin Chargaff 1950

LANDMARKS IN THE FIELD OF
MOLECULAR BIOLOGY
- Demonstrated in DNA, the # of
Adenine = Thymine; # of Cytosine =
Guanine
MILESTONE IN THE FIELD OF
MOLECULAR BIOLOGY
- James Dewey Watson, Francis Harry
Compton Crick (F. H. C. Crick) 1953
o Structure of DNA
o Proposed the double helical model
of DNA
o Based on the studies of Rosaline
Franklin (obtain images of DNA
using X-ray crystallography) and
Maurice Wilkins (idea)
- Watson, Crick and Wilkins
o Awarded noble price in 1962
o One of the most important
scientific discovery of 20th century.
- Matthew Meselson, Franklin
Kornberg 1957
o Confirmed Watson and Crick
semiconservative model of DNA
replication
- Arthur Kornberg 1958
o He isolated the first polymerizing
enzyme now known as dna
polymerase from E. Coli and got a
noble price 1959
- F. H. C. Crick 1958
o Originated the concept of the
central dogma of molecular
biology (describe the transfer of
genetic information into functional
macromolecules.
- Marshall Nirenberg, Heinrich
Matthaei 1961
o They crack the genetic code they
crack the genetic code present in
MRNA
- Francois Jacob, Jacques Monod 1961
o They put forward the operon
concept for regulation of gene
expression.
o Receive noble prize in 1965
- F. H. C. Crick 1966
o He proposed the Wobble
hypothesis
- Har Gobind Khorana, Marshall
Nirenberg, Robert Holley 1966
o They work out the complete
genetic code for one enzyme
o Receive noble prize in 1968
- Allan Maxam, Walter Gilbert 1977
o Describe the dna sequencing
technique
- Frederick Sanger with his team 1977
o Dna sequencing techniques (sanger
sequencing) noble price 1980
- Yoshinori Ohsumi 2016
 o Mechanisms for autophagy
(body’s way of cleaning up
damaged cells to regenerate newer
or healthier cells.
- William G. Kaelin Jr., Sir Peter J.
Ratcliffe, Gregg L. Semenza 2019
o How cells sense and adapt to
oxygen availability
CENTRAL DOGMA OF MOLECULAR
BIOLOGY
- Genetic material- substance that stores
information about structure function and
development of various characteristics of
a living organisms
o It is responsible for the transmission
of genetic information for all the
characteristics of living beings from
parents to progeny
o Following the rediscovery of
Mendel’s laws, geneticists where is
able to conclude that:
 Organisms characters are
controlled by genes.
 Genes are able to reproduce
themselves or replicate without
losing their information.
 Genes are arranged on the
chromosome in a linear function.
 Genes are transmitted from
parents to offspring generation
after generation almost unaltered.
o Major characteristics of genetic
material
 Storage of genetic information
(structure, function, development
and reproduction in all the living
cells of organism)
 Accurate replication (so that the
same genetic information as
present in parents cell are pass on
the daughter cells)
 Transcription
 Chemical stability (so that the
stored information are not loss)
 Variations (capable of undergo
modification and their
combination so that it can
contribute to adaptation leading to
evolution of living organisms)
CENTRAL DOGMA: DNA TO RNA TO
PROTEIN
1. The DNA produces exact copies of
itself by the process of replication as
it can only be synthesized from any
other molecules.
2. The genetic information and DNA is
transferred to RNA by the process of
transcription.
3. Then the RNA translates it to form
proteins by the process of
translation.
The MRNA burst the genetic code appear
from DNA translate it into the amino acid
sequences by polypeptide chain with the
help of TRNA and RRNA and synthesize
proteins.
Thus, the transfer of genetic information
from DNA to RNA and the proteins is called
the CENTRAL DOGMA OF
MOLECULAR BIOLOGY
**in later years it has been proved that the
DNA can enzymatically replicate itself by
DNA polymerase and RNA to DNA by
reverse transcriptase
CENTRAL DOGMA IN MOLECULR
BIOLOGY – the process by which the
instruction in DNA are converted into a
multifunctional product.
LESSON 2A:
DNA STRUCTURE AND
FUNCTION
DEOXYRIBONUCLEIC ACID (DNA)
 which contains the blueprint for
constructing a living organism, is the
center piece for research and clinical
analysis.
 DNA is the information molecule.
 It stores instructions for making other
large molecules, called proteins.
 These instructions are stored inside
each of your cells, distributed among
46 long structures called
chromosomes.
 These chromosomes are made up of
thousands of shorter segments of
DNA, called genes.
 Each gene stores the directions for
making protein fragments, whole
proteins, or multiple specific proteins.
 Molecular biology has historically traced
its beginnings to the first description of
the structure of DNA by James Watson
and Francis Crick in 1953.
 The description of the DNA structure
initiated the dramatic increase in the
knowledge of the biology and chemistry
of our genetic machinery.
 The impact of the Watson and Crick
discovery was so significant that it is
considered one of the most important
scientific discoveries of the 20th century.
 In 1909, Russian-American biochemist
Phoebus Levene

 identified the 5-carbon sugar ribose

as part of some nucleic acids

 In 1929, he discovered a similar

sugar— deoxyribose —in other
nucleic acids.

 He had revealed a major chemical

distinction between RNA and
DNA:

RNA has ribose, and DNA has

deoxyribose. Levene then
discovered that the three parts of
a nucleic acid, these are: 1. Five - carbon sugars
 Sugar
 Nitrogen-containing base
 Phosphorus containing  Nucleotide subunits of RNA contain a
component. pentose (five-carbon) sugar called
ribose.

 Nucleotide subunits of DNA contain

the sugar deoxyribose.

NOTE!
 The five carbon atoms in each pentose
sugar are assigned numbers 1’ through
5’.

 Primes are used in the numbering of the

ring positions in the sugars to
differentiate them from the ring positions
of the bases.

 Both sugars have an oxygen as a

member of the five-member ri
ng; the 5’carbon is outside the ring.
 The sugars differ only in the presence or are easily broken by enzymatic
absence (“deoxy”) of an oxygen in the hydrolysis.
2’position.

 This minor distinction between RNA
and DNA dramatically influences their
function.
 The remarkable versatility of RNA is
critically dependent on these 2’-hydroxyl
groups.
2. Nitrogenous bases
 As their name suggests, the bases are
nitrogen-containing molecules having the
chemical properties of a base (a
substance that accepts an H+ ion or
proton in solution).
NOTE!
 Two of the bases, adenine (A) and
guanine (G) have a double carbon–
nitrogen ring structure; these are called
purines.
 The negatively charged phosphates are
extremely insoluble in lipids, which
ensures the retention of nucleic acids
within the cell or nuclear membrane.
COMPOSITION OF DNA
 The DNA double helix is an icon for
modern biology; a form represented
from art galleries to corporate logos.
 The structure of DNA is certainly
aesthetically pleasing but is relatively
meaningless without an understanding
of how it relates to function.
 Likewise, function cannot truly be
understood without knowledge of
structure.

 The other three bases, thymine (T),

cytosine (C), and uracil (U) have a single
ring structure; these are called
pyrimidines.

3. The phosphate functional group

 The phosphate functional group (PO4)

gives DNA and RNA the property of an
acid (a substance that releases an H+ ion
or proton in solution) at physiological
pH, hence the name “nucleic acid.”

 The linking bonds that are formed from NUCLEOSIDES AND NUCLEOTIDES
phosphates are esters that have the
additional property of being stable yet

A DNA OR RNA CHAIN IS FORMED
IN A SERIES OF THREE STEPS:
1. In the first reaction, each base is
chemically linked to one molecule of
sugar at the 1’-carbon of the sugar,
forming a compound called a nucleoside.
2. When a phosphate group is also attached
to the 5’-carbon of the same sugar, the
nucleoside becomes a nucleotide.
3. Finally, nucleotides are joined
(polymerized) by condensation reactions
to form a chain. The hydroxyl group on
the 3’-carbon of a sugar of one
nucleotide forms an ester bond to the
phosphate of another nucleotide,
eliminating a molecule of water.
This chemical bond linking the sugar
components of adjacent nucleotides is
called a phosphodiester bond, or 5’ - 3’
phosphodiester bond, indicating the
polarity of the strand.
THE FOUR NUCLEOTIDE
MONOMERS ARE DISTINGUISHED
BY THEIR BASES. EACH TYPE OF
NUCLEOTIDE HAS A DIFFERENT
NUCLEOBASE STUCK TO ITS
DEOXYRIBOSE SUGAR.
 A nucleotide contains adenine.
 T nucleotide contains thymine.
 G nucleotide contains guanine.
 C nucleotide contains cytosine.
NOTE!
 All four of these nucleobases are
relatively complex molecules, with the
unifying feature that they all tend to have
multiple nitrogen atoms in their
structures.
 For this reason, nucleobases are often
also called nitrogenous bases.
SIGNIFICANCE OF 5’ AND 3’
 The ends of a DNA or RNA chain are
distinct and have different chemical
 properties. The two ends are designated
by the symbols 5’ and 3’.
 The symbol 5’refers to the carbon
in the sugar to which a phosphate
(PO4) functional group is attached.
oxygen) all the way to 5’ (“five
prime”), identifying the carbon that
sticks off the fourth and final carbon
in the deoxyribose ring.

 The symbol 3’ refers to the carbon

in the sugar ring to which a
hydroxyl (OH) functional group is
attached

NOTE!

 The asymmetry of the ends of a DNA

strand implies that each strand has a
polarity determined by which end
bears the 5′-phosphate and which end
bears the 3′-hydroxyl group.

 This 5′ → 3′ directionality of a
nucleic acid strand is an extremely
important property of the molecule.

 Understanding this directionality

(polarity) is critical for understanding
aspects of replication and
transcription, for reading a DNA
sequence, and for carrying out
experiments in the lab.

 In order to keep things organized,

biochemists have developed a
numbering system for talking about  A diagram showing the carbons on the
the molecular structure of nucleotides. ribose ring numbered.

 These numbers are applied to the  The phosphate group is attached to the 5'
carbon atoms in the sugar, starting at carbon. The -OH group is attached to the
the carbon immediately to the right of 3' carbon and the base is attached to the
the oxygen in the deoxyribose ring, 1' carbon.
and continuing in a clockwise fashion:
the numbers range from 1’ (“one
prime”), identifying the carbon
immediately to the right of the
 The phosphodiester bonds that join one
DNA nucleotide to another always link
the 3’ carbon of the first nucleotide to the
5’ carbon of the second nucleotide.
 This forms a covalent bond between the
oxygen sticking off the 3’ carbon of the
first nucleotide, and the phosphorous
atom in the phosphate group that sticks
off the 5’ carbon of the second
nucleotide. These bonds are called 3’-5’
phosphodiester bonds.
 Each time nucleotides are bound
together, a water molecule is removed (or
“lost”) through a process
called dehydration synthesis.  and a newly synthesized strand oriented
 Many molecules rely on dehydration
synthesis to assist with forming
polymers.
LESSON 2B:
DNA REPLICATION
 DNA synthesis proceeds in the 5 to 3
direction, DNA polymerase, the enzyme
responsible for polymerizing the
nucleotides, uses a guide, or template, to
determine which nucleotides to add.
 The enzyme reads the template in the 3
to 5 direction.
 The resulting double strand, then, will
have a parent strand in one orientation
in the opposite orientation.
 Each cell division cell must copy its
entire DNA.
 So each daughter cell gets a complete
copy
AT FIRST, SOME RESEARCHERS
SUGGESTED THAT DNA MIGHT
REPLICATE IN ANY OF THREE
POSSIBLE WAYS:
1. Semiconservative
2. conservative, with one double helix
specifying creation of a second double
helix, and
3. dispersive, with a double helix
shattering into pieces that would join
with newly synthesized DNA pieces
to form two molecules.

SEMICONSERVATIVE
REPLICATION
 Predicted by Watson and Crick; It is the
key to maintain the sequence of the
nucleotides in DNA through new
generations.
 Every cell in a multicellular organism or
in a clonal population of unicellular
organisms carries the same genetic
information.
 It is important that this information, in
the form of the DNA sequence, be
transferred faithfully at every cell
division.
 The replication apparatus is designed to
copy the DNA strands in an orderly way
with minimal errors before each cell
division.
 A few years after solution of the double
helix, the mechanism of
semiconservative replication was
demonstrated by Matthew Meselson and
Franklin Stahl using the technique of
equilibrium density centrifugation on a
cesium gradient.
 The order of nucleotides is maintained
because each strand of the parent double
helix is the template for a newly
replicated strand.
 Human DNA replicates about 50 bases
per second.
 To get the job done, a human
chromosome replicates simultaneously at
hundreds of points along its length, and
the pieces join.
 A site where DNA is locally opened
resembling a fork, is called a replication
fork.
Note!
Ligase comes from the Latin word meaning
“to tie”.
 This replication process can be observed
by ELECTRON MICROSCOPY as a
FORKED STRUCTURE OR
REPLICATION FORK.
 OKAZAKI FRAGMENT, small pieces
of DNA about 1000 bases in length.
DNA REPLICATION PROCESS
1. DNA replication occurs during S
PHASE of the cell cycle.
2. When DNA replicates, it unwinds,
breaks, builds a new nucleotide chain,
and mends Enzymes called helicases
unwind and hold apart replicating DNA,
enabling other enzymes to guide the
assembly of a new DNA strand.
3. DNA replication begins when a helicase
breaks the hydrogen bonds that connect a
base pair. Binding proteins hold the two
strands apart.
Another enzyme, primase, then attracts
complementary RNA nucleotides to
build a short piece of RNA, called an
RNA primer, at the start of each segment
of DNA to be replicated.
 The RNA primer is required because the
major replication enzyme, DNA 9. DNA polymerase also “proofreads” as it
polymerase (DNAP), can only add bases goes, excising mismatched bases and
to an existing nucleic acid strand. inserting correct ones.

4. The RNA primer attracts DNAP, which
brings in DNA nucleotides
complementary to the exposed bases on
the parental strand; this strand serves as a
mold, or template. New bases are added
one at a time, starting at the RNA primer.
It also removes the RNA primer and
replaces it with the correct DNA bases.
Yet another enzyme, called an annealing
helicase, rewinds any sections of the
DNA molecule that remain unwound.

5. The new DNA strand grows as hydrogen 10.Finally, ligases seal the entire sugar-
bonds form between the complementary phosphate backbone.
bases. The nucleotides are abundant in
cells and are synthesized from dietary
nutrients.

6. DNAP works directionally, adding new

nucleotides to the exposed 3 ′ end of the
sugar in the growing strand.

Overall, replication proceeds in a 5 ′ to 3

′ direction, because this is the only
chemical configuration in which DNAP
can add bases.

7. Next, an enzyme called a ligase then

seals the sugar- phosphate backbones of
the pieces, building the new strand.
These pieces, up to 150 nucleotides long,
are called Okazaki fragments, after their
discoverer.

8. DNA polymerase also “proofreads” as it

goes, excising mismatched bases and
inserting correct ones. It also removes the
RNA primer and replaces it with the
correct DNA bases. LESSON 2C:
ENZYMES THAT
Yet another enzyme, called an annealing
helicase, rewinds any sections of the METABOLIZE DNA
DNA molecule that remain unwound.
RESTRICTION ENZYMES
 These are endonucleases that recognize
specific base sequences and break or
restrict the DNA polymer at the sugar-
phosphate backbone.
 These enzymes were originally isolated
from bacteria where they function as part
of a primitive defense system to cleave
foreign DNA entering the bacterial cell.
 The ability of the cell to recognize
foreign DNA depended on both DNA
sequence recognition and methylation.
 Restriction enzymes are named for the
organism from which they were isolated.
 Example, BamHI was isolated from
Bacillus amyloliquefaciens
TYPE I RESTRICTION ENZYMES
 Have both nuclease and methylase
activity in a single enzyme.
 They bind to host-specific DNA sites of
4–6 bp separated by 6–8 bp and
containing methylated adenines.
 The site of cleavage of the DNA
substrate can be over 1000 bp from this
binding site.
 An example of a type I enzyme is EcoK
from E. coli K 12. It recognizes the site:
5-ACNNNNNNGTGCTGNN
N N N N C A C G - 5 where N
represents nonspecific nucleotides and
the adenine residues (A) are methylated.
TYPE II RESTRICTION ENZYMES
 Are those used most frequently in the
laboratory.
 These enzymes do not have inherent
methylation activity in the same
molecule as the nuclease activity.
 They bind as simple dimers to their
symmetrical DNA recognition sites.
 These sites are palindromic in nature;
that is, they read the same 5 to 3 on both
strands of the DNA, referred to as
bilateral symmetry.
TYPE III RESTRICTION ENZYMES
 Resemble type I enzymes in their ability
to both methylate and restrict (cut) DNA.
 Like type I, they are complex enzymes
with two subunits.
 Recognition sites for these enzymes are
asymmetrical, and the cleavage of the
substrate DNA occurs 24–26 bp from the
site to the 3 side.
 An example of a type III enzyme is  degrades single-stranded DNA from the
HinfIII from H. influenzae. 3 hydroxyl end, producing
mononucleotides.
 Its activity is optimal on long single-
 It recognizes the site: 5 - C G A A T G C stranded ends, slowing significantly as it
T T A - 5 where the adenine methylation approaches a double-stranded region.
occurs on only one strand.
EXONUCLEASE III FROM E. COLI

DNA LIGASE  removes 5 mononucleotides from the 3

end of double- stranded DNA in the
 catalyzes the formation of a presence of Mg2 and Mn.
phosphodiester bond between adjacent 3-
hydroxyl and 5-phosphoryl nucleotide  It also has some endonuclease activity,
ends. Its existence was predicted by the cutting DNA at apurinic sites.
observation of replication,
recombination, and repair activities in  Exo III removes nucleotides from blunt
vivo. ends, recessed ends, and nicks, but will
not digest 3 overhangs.

 Exo III has been used in the research

setting to create nested deletions in
double-stranded DNA or to produce
single- stranded DNA for dideoxy
sequencing.

METABOLIZING ENZYMES
EXONUCLEASE VII FROM E. COLI

EXONUCLEASES
 digests single-stranded DNA from either
the 5 phosphate or 3 hydroxyl end.
 Degrade DNA from free 3 hydroxyl or 5
phosphate ends.  It is one of the few enzymes with 5
exonuclease activity.
EXONUCLEASE I FROM E. COLI
 Exo VII can be employed to remove long
single strands protruding from double-
stranded DNA.
NUCLEASE Bal31 FROM Alteromonas
espejiani
 can degrade single- and double-stranded
DNA from both ends.
 Because its activity at 20oC is slow
enough to control with good resolution, it
has been used extensively in research
applications to make nested deletions in
DNA.
S1 NUCLEASE FROM Aspergillus
oryzae
 It hydrolyzes single-stranded DNA or
RNA into 5 mononucleotides.
 It also has endonuclease capability to
hydrolyze single-stranded regions such
as gaps and loops in duplex DNA.
 It was used extensively in early RNAse
protection assays of gene expression. It is
also used for nuclease mapping
techniques.
recBC NUCLEASE FROM E. coli
 It digests DNA from either the 3
hydroxyl or the 5 phosphate ends.
 It has some endonuclease activity on
duplex DNA, generating short fragments,
or oligonucleotides.
MICROCOCCAL NUCLEASE
 Digests single- and doublestranded DNA
and RNA at AT- or AU-rich regions.
 Although this enzyme can digest duplex
DNA, it prefers single-stranded
substrates.
 It is used in the laboratory to remove
nucleic acid from crude extracts and also
for analysis of chromatin structure.
DEOXYRIBONUCLEASE I (DNAse I)
FROM bovine pancreas
 Digests single-and double-stranded DNA
at pyrimidines to
oligodeoxyribonucleotides; so,
technically, it is an endonuclease.
 It is used in both research and clinical
laboratories to remove DNA from RNA
preparations.
 DNAse I has also been used to detect
exposed regions of DNA in DNA protein
binding experiments.
HELICASES
 Release of DNA for transcription,
replication, and recombination without
 tangling is brought about through cutting for the specific methylation patterns
and reclosing of the DNA sugar- in differentiated cells
phosphate backbone.

 These functions are carried out by a

series of enzymes called helicases.

METHYLTRANSFERASES

 catalyze the addition of methyl groups

to nitrogen bases, usually cytosine in
DNA strands.

 Most prokaryotic DNA is methylated,

or hemimethylated (methylated on
one strand of the double helix and not
the other), as a means to differentiate
host DNA from nonhost and to
provide resistance to restriction
enzymes.

 Unlike prokaryotic DNA, eukaryotic

DNA is methylated in specific
regions.
 In eukaryotes, DNA binding proteins
may limit accessibility or guide
methyltransferases to specific regions
of the DNA.
TWO TYPES OF
METHYLTRANSFERASE
1. Maintenance Methyltransferase
 work throughout the life of the cell
and methylate hemimethylated DNA.
2. De nove Methyltransferase
 work only during embryonic
development and may be responsible
LESSON 3A:
FUNDAMENTALS OF
RIBONUCLEIC ACID
BIOCHEMISTRY
RNA TYPES AND STRUCTURES
 It is a polymer of nucleotides similar to
DNA.
 It differs from DNA in the sugar
moieties, having ribose instead of
deoxyribose and, in one nitrogen base
component, having uracil instead of
thymine (thymine is 5-methyl uracil).
 Furthermore, RNA is synthesized as a
single strand rather than as a double
helix.
 Although RNA strands do not have
complementary partner strands, they are
not completely single-stranded.
 Through internal homologies, RNA
species fold and loop upon themselves to
take on as much of a double-stranded
character as possible.
 RNA can also pair with complementary
single strands of DNA or RNA and form
a double helix.
 There are several types of RNAs found in
the cell.
 Ribosomal RNA, messenger RNA,
transfer RNA, and small nuclear RNAs
have distinct cellular functions.
 RNA is copied, or transcribed, from
DNA.
 Carries the genetic information from
DNA and is used as a template for
protein synthesis.
 Most mRNAs are 500 to 4,500 bases
long.
 Each three mRNA bases in a row form a
genetic code word, or codon.
 Polycistronic – coding more than one
protein on the same mRNA; most
prokaryotes are Polycistronic.
 Monocistronic – coding one protein on
the same mRNA; most Eukaryotes are
monocistronic.

DIFFERENCES OF DNA AND RNA

MESSENGER RNA PROCESSING
DNA RNA
• Double Stranded Single strands 1. Polyadenylation
• Base- Thymine • Base- Uracil
• Sugar- Deoxyribose • Sugar- Ribose  Study of mRNA in eukaryotes was
• Cannot function as • Can function as facilitated by the discovery that most
enzyme enzyme messengers carry a sequence of
polyadenylic acid at the 3-terminus, the
 In the synthesis of protein there three poly(A) tail.
types of RNA that participate and that  The run of adenines was first discovered
play different roles: by hydrogen bonding of mRNA to
polydeoxythymine on poly(dT) cellulose.
 4 Polyuridine or polythymine residues
covalently attached to cellulose or
I. MESSENGER RNA (mRNA) sepharose substrates are often used to
specifically isolate mRNA in the
laboratory.
 The poly(A) tail is not coded in genomic
DNA.
 It is added to the RNA after synthesis of
the pre-mRNA.
 A protein complex recognizes the RNA
sequence, AAUAAA, and cleaves the
RNA chain 11–30 bases 3 to that site.
 The enzyme that cuts pre-mRNA in
advance of polyadenylation has not been
identified. Recent studies suggest that a
component of the protein complex
related to the system that is responsible
for removing the 3 extension from pre-
transfer RNAs may also be involved in
generation of the 3 ends on mRNA.
 The enzyme polyadenylate polymerase is
responsible for adding the adenines to the
end of the transcript.
 A run of up to 200 nucleotides of
poly(A) is typically found on mRNA in
mammalian cells.
2. Capping
 Capping occurs after initiation of
transcription, catalyzed by the enzyme
guanylyl transferase.
 Eukaryotic mRNA is blocked at the 5-
terminus by an unusual 5-5
pyrophosphate bridge to a methylated
guanosine.
 The structure is called a CAP. The cap is
a 5-5 pyrophosphate linkage of 7-methyl
guanosine to either 2 O-methyl guanine
or 2 O-methyl adenine of the mRNA, 7-
methylG5 ppp 5 G or A 2 O-methyl
pNpNpNp where p represents a
phosphate group, N represents any
nucleotide.
 The cap confers a protective function as
well as serves as a recognition signal for
the translational apparatus.
 Caps differ with respect to the
methylation of the end nucleotide of the
mRNA.
 In some cases, 2O-methylation occurs
not only on the first but also on the
second nucleotide from the cap.
 Other caps methylate the first three
nucleotides of the RNA molecule.
3. Splicing
 Prokaryotic structural genes contain
uninterrupted lengths of open reading
frame, sequences that code for amino
acids.
 In contrast, eukaryotic coding regions are
interrupted with long stretches of
noncoding DNA sequences called
introns.
 Newly transcribed mRNA, heteronuclear
RNA (hnRNA), is much larger than
mature mRNA because it still contains
the intervening sequences.
 Labeling studies demonstrated that the
hnRNA is capped and tailed and that
these modifications survive the transition
from hnRNA to mRNA, which is simply
a process of removing the intervening
sequences from the hnRNA.
 Introns are removed from hnRNA by
splicing.
 The remaining sequences that code for
the protein product are exons.
 Splicing may be important for timing of
translation of mRNA in the cytoplasm,
although it is not necessarily required as
cloned genes synthesized in vitro without
introns are expressed in eukaryotic cells.
 Introns may have evolved as a means of
increasing recombination frequency
within genes as well as between genes.

 The discontinuous nature of eukaryotic
genes may also protect the coding
regions from genetic damage by toxins or
radiation.
II. RIBOSOMAL RNA (rRNA)
 Major constituent of the cellular particles
called ribosomes on which protein
synthesis takes place.
 It compromises 80-90% of the total
cellular RNA.
 Sedimentation coefficient (S)
 Various types of ribosomal RNA are
named for their sedimentation coefficient
in density gradient centrifugation.
 (rRNA) molecules range from 100 to
nearly 3,000 nucleotides long.
 In prokaryotes there are three rRNA
species, these are:
A. 16S- found in the ribosome small subunit
B. 23S- found in the ribosome large subunit
C. 5S- found in the ribosome large subunit
 In eukaryotes, rRNA is copied from
DNA as a single 45S precursor RNA
that is highly processed into:
A. 18S- found in the ribosome small subunit
B. 5.8S-found in the large subunit
C. 28S- found in the large subunit
III. TRANSFER RNA (tRNA)
 Incorporates a particular amino acid
subunit into the growing protein when it
recognizes a specific group of three
adjacent bases in the mRNA.
 Binds an mRNA codon at one end and a
specific amino acid at the other.
 A tRNA molecule is only 75 to 80
nucleotides long. Some of its bases form
weak chemical bonds with each other,
folding the tRNA into loops in a
characteristic cloverleaf shape.
 One loop of the tRNA has three bases in
a row that form the anticodon, which is
complementary to an mRNA codon.
 The end of the tRNA opposite the
anticodon strongly bonds to a specific
amino acid.
 A tRNA with a particular anticodon
sequence always carries the same amino
acid.
 For example, a tRNA with the anticodon
sequence GAA always picks up the
amino acid phenylalanine.
 Enzymes attach amino acids to tRNAs
that bear the appropriate anticodons,
where they form chemical bonds.
MICRO RNAs (miRNA)
 These are tiny regulatory RNAs; 21-25 nt
in length.
 Derived from endogenous RNA hairpin
structures.
 Caenorhabditis elegans- the worm in
which the micro-RNA was discovered.
SMALL NUCLEAR RNA
 Another type of cellular RNA is the
small nuclear RNA (snRNA), which
functions in splicing (removal of introns
from freshly transcribed RNA) in
eukaryotes. Small nuclear RNA stays in
the nucleus after its transcription by
RNA polymerase I or III.
 Small nuclear RNAs from eukaryotic
cells sediment in a range of 6-8S.
 Small nuclear RNAs serve mostly a
structural role in the processing of
mRNA.
OTHER SMALL RNAs
 Since the late 1990s a growing variety of
small RNAs (sRNA) have been described
in prokaryotes and eukaryotes, including
tiny noncoding RNAs (tncRNA, 20- 22
b), small modulatory RNA (smRNA, 21-
23b), small nucleolar RNAs (snoRNA),
tmRNA26 and others.
 In addition to RNA synthesis and LESSON 3B:
processing, these molecules influence RNA MATABOLISM
numerous cellular processes, including
plasmid replication, bacteriophage ENZYMES AND
development, chromosome structure, and REGULATION OF
development. TRANSCRIPTION TYPES
 These small untranslated RNA
molecules have been termed sRNAs in
AND STRUCTURES
bacteria and noncoding RNAs (ncRNAs)
in eukaryotes.
RNA METABOLIZING ENZYMES
4
RNA POLYMERASE
 Catalyzes RNA synthesis.
 One multi-subunit prokaryotic enzyme is
responsible for the synthesis of all types
of RNA in the prokaryotic cell.
 Eukaryotes have three different RNA
polymerase enzymes.

1. DNA - dependent RNA polymerases

require a DNA template.
2. RNA - dependent RNA polymerases
require an RNA template.

 In prokaryotes, all types of RNA are

synthesized by a single RNA
polymerase.

RIBONUCLEASES

 Ribonucleases degrade RNA in a manner

similar to the degradation of DNA by
deoxyribonucleases.
 An endoribonuclease, cleavage, and
polyadenylation specific factor (CPSF)
 are required for proper termination of
RNA synthesis.
 32,33 Along with RNA polymerase II
subunits and other proteins, this enzyme
cuts the nascent RNA transcript before
addition of the polyA tail by poly A
polymerase.
RNA HELICASES
 RNA helicases catalyze the unwinding of
double stranded RNA.
 RNA synthesis and processing require
the activity of helicases.
 These enzymes have been characterized
in prokaryotic and eukaryotic organisms.
 Some RNA helicases work exclusively
on RNA. Others can work on DNA:RNA
heteroduplexes and DNA substrates.
 Evidence suggests that transcription
takes place at discreet stations of the
nucleus into which the DNA molecules
move. One of these sites, the nucleolus,
is the location of ribosomal RNA
synthesis.
REGULATION OF TRANSCRIPTION
Transcription Factors
 1961- when French biologists François
Jacob and Jacques Monod described
the remarkable ability of E. coli bacteria
to produce the enzymes to metabolize the
sugar lactose—but only when lactose is
in the cell’s surroundings. Jacob and
Monod discovered that a modified form
of lactose “turned on” the genes whose
encoded proteins break it down.
 To manage this, groups of proteins called
transcription factors come together,
forming an apparatus that binds DNA at
certain sequences and initiates
transcription at specific sites on
chromosomes.
 Transcription factors include regions
called binding domains that guide them
to the genes they control. The DNA
binding domains have very colorful
names, such as “helix turn-helix,” “zinc
fingers,” and “leucine zippers,” that
reflect their distinctive shapes.
Two types of factors are responsible for
regulation of RNA synthesis:
 cis factors - are DNA sequences that
mark places on the DNA involved in
the initiation and control of RNA
synthesis.
=For transcription to occur, several proteins
must assemble at the gene’s transcription
initiation site including specific and general
transcription factors and the RNA
polymerase complex.
 trans factors- are proteins that bind to
the cis sequences and direct the assembly
of transcription complexes at the proper
gene.
Steps of Transcription
Transcription is described in three steps:
1. Initiation
2. Elongation
3. Termination
5
Transcription Initiation
 Transcription factors and RNA
polymerase are attracted to a promoter,
which is a special sequence that signals
the start of the gene.
 The first transcription factor to bind,
called a TATA binding protein, is
chemically attracted to a DNA sequence
called a TATA box—the base sequence
TATA surrounded by long stretches of G
and C.
 Once the first transcription factor binds,
it attracts others in groups. Finally, RNA
polymerase joins the complex, binding
just in front of the start of the gene
sequence.
Setting the Stage for Transcription to begin:
1. Proteins that initiate transcription
recognize specific sequences in the promoter
region of a gene.
2. A binding protein recognizes the TATA
region and binds to the DNA. This allows
other transcription factors to bind.
3. The bound transcription factors form a
pocket that allows RNA polymerase to bind
and begin making RNA.
Transcription Elongation
 Enzymes unwind the DNA double helix
locally, and free RNA nucleotides bond
with exposed complementary bases on
the DNA template strand.
 RNA polymerase adds the RNA
nucleotides in the sequence the DNA
specifies, moving along the DNA strand
in a 3 ′ to 5 ′ direction, synthesizing the
RNA molecule in a 5 ′ to 3 ′ direction.
 A terminator sequence in the DNA
indicates where the gene’s RNA-
encoding region ends.
 When this spot is reached, the third stage,
transcription termination, occurs.
➢ A typical rate of transcription in humans
is 20 bases per second.
Transcription Termination
 It is the ending of transcription and
occurs when RNA polymerase crosses a
stop sequence in the gene. The mRNA
strand is complete, and it detaches from
DNA.
Two Major Termination Strategies
Found in Bacteria:
1. Rho-dependent termination
2. Rho-independent termination
Rho-dependent Termination
 In Rho-dependent termination, the
RNA contains a binding site for a
protein called Rho factor.
 Rho factor binds to this sequence and
starts “climbing” up the transcript
towards RNA polymerase.
 When it catches up with the
polymerase at the transcription
bubble, Rho pulls the RNA transcript
and the template DNA strand apart,
releasing the RNA molecule and
ending transcription.
 Another sequence found later in the
DNA, called the transcription stop
point, causes RNA polymerase to
pause and thus helps Rho catch up.
Rho-independent Termination
 Rho-independent termination depends on
specific sequences in the DNA template
strand.
 As the RNA polymerase approaches the
end of the gene being transcribed, it hits
a region rich in C and G nucleotides.
 The RNA transcribed from this region
folds back on itself, and the
complementary C and G nucleotides bind
together.
 The result is a stable hairpin that causes
the polymerase to stall.
Getting Started: Cuvettes and Solutions
To begin using, you have to have your
samples ready,
Calibration
SPECTOPHOTOMETER
Parts of Spectrophotometer
 Sample Compartment – the led
opens and closes to insert sample
inside
 Wavelength Control Knob – to
change wavelength
 Zero Control Knob – left
 Transmittance/Absorbance Control
Knob – right
 Mode select button – transmittance,
absorbance, concentration, factor
1. Prepare a blank solution, the blank
solution has everything in it except
for the molecule or compound you
wish to measure.
2. You should have a tube which
contains a sample which you want to
measure.
When taking readings, its best to use the
transmittance. (Select “Transmittance”
before making adjustments)
1. Use the wavelength control knob to
select the desired wavelength.
2. Use the Zero Control Knob to set the
transmittance to 0.0%
3. Open the sample compartment
4. Put the blank solution cuvette in
(always wipe the button of cuvette
before inserting to make sure nothing
interferes the reading). Notice the
vertical line on the cuvette, match up
this line with the mark at the front
edge of the sample compartment.
 5. Use the transmittance/absorbance
control Knob to set the Transmittance
to 100.0%
Taking measurements
- Once you finish the calibration, you can
take the Blank Solution cuvette out.
- Now you can put the Sample Solution
cuvette in to take a measurement. Be sure
to align the marks.
- Push mode select to read absorbance
- Only use transmittance and absorbance
- When you remove your sample, the
transmittance should return to 0.0%
- You can take as many readings at the
same wavelength as you want by simply
changing the Sample cuvettes.
*** Recalibrate every time you change the
wavelength.
LABORATORY:
SPECTROPHOTOMETRY
- Used to determine the quantity of a
specific molecule present in a solution.
How does it work?
- It’s function depends on the physical
properties of the molecule being
measured.
- Visible light of a specific wavelength is
passed through a solution inside the
machine.
- The light strikes molecules in the
solution:
 Some of the light is absorbed by
those molecules
 Some of the light is reflected or not
absorbed.
The spectrophotometry directly measures
how much of the light passed through a
sample and it’s either absorb or
transmitted (not absorbed).
Visible light
Light is a form of radiant energy. There has
to be a source of radiation In order for us to
perceive it. The sun, light bulbs, LEDs,
lasers, etc. are all examples of common light
sources. Each color of light is radiation of a
specific wavelength.
Electeomagnetic Spectrum
A visible light is a very tiny fraction of the
entire spectrum of radiation.
Normal measurement 400-750nm
- Less than 400nm is considered as
“ultraviolet” radiation.
- More than 750nm is considered as
“infrared” radiation.
- Energy increases as wavelength
decreases.
Color
White light is a combination of all colors of
light.
Effect of a “prism” or diffraction grating on
white light is:
- Each wavelength of light will travel
through the prism at a different angle
- Each wavelength of light will exit the
prism separated from each other.
- “Rainbow”
The color that something appears depends
on what pigments they contain.
Pigments are molecules that absorb specific
wavelength of visible light.
A red flower appears only red because it
absorbs all colors of light except for red.
Thus, Its pigment strongly absorbs All other
colors of light that reflects red light.
***Thus whatever color something
appears to us that is the color it absorbs
the least.
Using prism, one specific wavelength
(color) of light can be passed trough
sample.
 Percent Transmittance:
The amount of that wavelength of light that
passes through is measured directly. Scales
ranges form 0% - 100%.
 Absorbance:
The amount of light absorbed by the sample
( calculated based on % transmittance).
Scales ranges from 0 to infinite.
(Absorbance has no units)
***High % of transmittance = Low
absorbance and vice versa
FACTORS THAT AFFECT
SPECTROPHOTOMETER
MEASUREMENTS
1. Colored Molecules – the molecules must
have some kind of color to work. If a
molecule does not strongly absorb any
wavelength of visible light it will appear
clear in solution. It can not be
measured…
Unless we add an “indicator”
molecule.
Indicator change colors upon reaction
with a colorless molecule.
The indicator can allow us to
indirectly obtain a measurement.
2. Concentration – there must not be too
much or too little of the molecule in the
sample. The spectrophotometer has
detection limits.
3. Light Path - the distance the light passes
through a sample affect the
 measurement. Use a cuvette with specific
diameter or thickness.
4. Calibration - you must calibrate the
spectrophotometer using a blank solution
reference before taking measurements.
 A blank contains everything that is in
your sample tube except the molecule
you are measuring.
 You must recalibrate the
spectrophotometer every time you set
it to a different wavelengths.
 Detection limit of the
spectrophotometer - you can not
obtain readings at concentrations that
are too low or too high.
 You may have to calculate the
absorbance at very low %
transmittance readings.
 You may have to dilute your sample
further if it is too concentrated to
measure.
SPECTROPHOTOMETER ANALYSIS
To measure the concentration of a specific
molecule:
1. Create an observance profile of that
molecule.
 Make a solution containing a known
concentration of the molecule of
interest.
 The solution should be only
slightly tinted in color.
 Calibrate the spectrophotometer
using a proper blank solution
 Measure the % transmittance and
absorbance of this one known
solution at different wavelengths
between 400 and 750 nm
 Start by measuring in 100nm
increments: (400, 500, 600,
700nm)
 Find the two wavelengths with
the highest absorbance values
 Measure in smaller increments
between those two wavelengths
 Example: if 600 and 700nm are
the highest, also take readings at
620, 640, 660 and 680nm
2. Create a standard curve
 Make a series of solutions (5 or
more)
 Each solution containing a known
concentration of the molecule of interest
 Each solution of a different concentration
ranging from low to high
 Make each concentration a fraction of the
max (high) concentration
 Example : if your high concentration is
0.10mg/mL then make series of known
solutions containing 0.02, 0.04, 0.06,
0.08 and 0.10 mg/mL
 Use your absorbance profile to select the
best wavelength to measure at calibrate
the spectrophotometer using a proper
blank solution measure the %
transmittance and absorbance of each
known concentration.
 Create a line graph of absorbance vs.
Concentration.
*** use this data to determine the
concentration of each sample.
3. Set the spectrophotometer to the
optimum absorbance wavelength from
your absorbance profile.
4. Measure the observance of your sample
5. Determine the concentration of each An absorbance spectrum is the plot of the
sample using standard curve. absorbance vs the wavelength of the incident
light.
SPECTROPHOTOMETRY

THE BEER-LAMBERT’S LAW

 Spectroscopy is the study of

electromagnetic radiation emitted or
absorbed by a given chemical species.
 Spectrophotometer an instrument that
measures the amount of light absorbed
by a substance. A transmittance spectrum is the plot of the
transmittance or %T versus the wavelength
l: the distance the light travels to the of the incident light.
solution

I0: intensity of incident light

Beer-Lambert Law
I: intensity of the light after it has passed
through the sample. The absorbance can be expressed by the
beer lambert law.
Transmittance (T) is a measure of fraction of
light that passes through the sample. It is the A=Ex1xc
ratio between I and I0 A : is the absorbance of solution (no unit)
T = I/I0 E : is the molar absorptivity or the molar
The percent transmittance (%T) could also extinction coefficient (in L/mol.cm),
be used : %T = T x 100 l : is the distance the light travels through
The absorbance (A) is the amount of light the solution (in cm)
absorbed by the sample. c : is the concentration of absorbing species
A = -log T = -log I/I0 (in mol/L)

The absorbance can also be given in terms

of the percentage transmittance. ELECTROPHORESIS
A = 2 -log (%T) Electrophoresis is the movement of
molecules by an electric current. This can
occur in solution, but it is practically done in
ABSORBANCE SPECTRUM a matrix to limit migration and contain the
migrating material. Electrophoresis is
routinely applied to the analysis of proteins
and nucleic acids. Each phosphate group on
a DNA polymer is ionized, making DNA a
negatively charged molecule. Under an
electric current, DNA will migrate toward
the positive pole (anode). When DNA is
applied to a macromolecular cage such as
agarose or polyacrylamide, its migration
under the pull of the current is impeded,
depending on the size of the DNA and the
spaces in the gel. Because each nucleotide
has one negative charge, the charge-to-mass
ratio of molecules of different sizes will
remain constant. DNA fragments will
therefore migrate at speeds inversely related
to their size. Electrophoresis can be
performed in tubes, slab gels, or capillaries.
Slab gel electrophoresis can have either a
horizontal or vertical format.
GEL SYSTEM
 Gel matrices provide resistance to the
movement of molecules under the force
of the electric current.
 They prevent diffusion and reduce
convection currents so that the separated
molecules form a defined group, or
“band.”
 The gel can then serve as a support
medium for analysis of the separated
components.
 These matrices must be unaffected by
electrophoresis, simple to prepare and
amenable to modification.
 Agarose and polyacrylamide are
polymers that meet these criteria.
AGAROSE GEL
 Agarose is a polysaccharide polymer
extracted from seaweed.
 It is a component of agar used in
bacterial culture dishes.
 Agarose is a linear polymer of
agarobiose, which consists of 1,3-
linked--D-galactopyranose and 1,4linked
3,6-anhydro--L-galactopyranose.
 Hydrated agarose gels in various
concentrations, buffers, and sizes can be
purchased ready for use.
 Alternatively, agarose can be purchased
and stored in the laboratory in powdered
form.
 For use, powdered agarose is suspended
in buffer, heated, and poured into a mold.
 The concentration of the agarose dictates
the size of the spaces in the gel and will,
therefore, be determined by the size of
DNA to be resolved (Table 5.1).
 Small pieces of DNA (50–500 bp) are
resolved on more concentrated agarose
gels, e.g., 2%–3% (Fig. 5-3).
 Larger fragments of DNA (2000–50,000)
are best resolved in lower agarose
concentrations, e.g., 0.5%–1%.
 Agarose concentrations above 5% and
below 0.5% are not practical.
 High-concentration agarose will impede
migration, whereas very low
concentrations produce a weak gel with
limited integrity.
 Double-stranded DNA and RNA are  This slows and distorts the migration of
analyzed by native gel electrophoresis. the samples, reducing resolution and
 The relationship between size and speed smearing the bands.
of migration can be improved by
separating single-stranded nucleic acids;
however, both DNA and RNA favor the
double-stranded state.
 Unpaired, or denatured, DNA and RNA
must, therefore, be analyzed in
conditions that prevent the hydrogen
bonding between complementary
sequences.
 These conditions are maintained through
a combination of formamide mixed with
the sample, urea mixed with the gel,
and/or heat-denaturing electrophoresis.
 The physical characteristics of the
agarose gel can be modified by altering
its polymer length and helical
parameters.
 Several types of agarose are thus
available for specific applications.

 The resolving properties differ in these

preparations as well as the gelling
properties.

 Low-melting agarose is often used for re-

isolating resolved fragments from the
gel. Other agarose types give better
resolution of larger or smaller fragments.

 Modern agarose preparations are

sufficiently pure to avoid problems such
as electroendosmosis, a solvent flow
toward one of the electrodes, usually the
cathode (negative), in opposition to the
DNA or RNA migration.