Module 2

Biochemistry and molecular aspects of life

• Biochemistry, study of the chemical substances and processes that occur in plants, animals, and
microorganisms and of the changes they undergo during development and life.
• The living matter is composed of mainly six elements — carbon, hydrogen, oxygen, nitrogen,
phosphorus and sulphur.

Carbohydrates

• Carbohydrates are the most abundant organic molecules in nature. They are primarily composed
of the elements carbon, hydrogen and oxygen.
• The name carbohydrate literally means ‘hydrates of carbon.’ Some of the carbohydrates possess
the empirical formula (CH2O)n . However this is not true in case of all the carbohydrates.
• Carbohydrates may be defined as polyhydroxy- aldehydes or ketones or compounds which
produce them on hydrolysis. The term ‘sugar’ is applied to carbohydrates soluble in water and
sweet to taste.

Classification of Carbohydrates:

They are broadly classified into 3 groups—monosaccharides, oligosaccharides and polysaccharides.

This categorization is based on the number of sugar units. Mono- and oligosaccharides are sweet to
taste, crystalline in character and soluble in water, hence they are commonly known as sugars.

Monosaccharides

• Monosaccharides are the simplest sugars which cannot be further hydrolysed.

• Monosaccharides are regarded as trioses (3C), tetroses (4C), pentoses (5C), hexoses (6C) and
heptoses (7C).
1. Triose- Glyceraldehyde
2. Tetrose- Erythrose
3. Pentose- Ribose, Deoxyribose
4. Hexose- Glucose, Fructose, Galactose
5. Heptose- Sedoheptulose
• When a monosaccharide has aldehydic functional group they are called aldoses eg – Glucose.
When a carbohydrate has ketonic group, they are called ketoses eg- fructose.

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Disaccharide

• A disaccharide is formed when glycosidic bond forms between two monosaccharide units.
Eg- Sucrose: Glucose + Fructose
Lactose: Glucose + Galactose
Maltose: Glucose + Glucose

Oligosaccharide

• An oligosaccharide is made by joining of 3 or more monosaccharide units.

Example: Raffinose- Glucose + Galactose + Fructose
Stachyose- Glucose + Galactose + Galactose + Fructose

Polysaccharide

• A polysaccharide is made of several thousands of monosaccharide units.

Eg- Cellulose- Present in cell wall of plants
Starch- Stored food material in plants
Glycogen- Stored food material in animals

Proteins

• Proteins are the polymers of amino acids. Proteins are formed by the formation of peptide bond.
• There are as many as 300 different amino acids but only 20 are known as standard amino acids
and occur repeatedly in structure of protein
• Amino acids are a group of organic compounds containing two functional groupsamino and
carboxyl. The amino group (-NH2) is basic while the carboxyl group (-COOH) is acidic in
nature.
• Proteins are the polymers of amino acids. The structure of proteins is rather complex which can
be divided into 4 levels of organization.

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 1. Primary structure: The linear sequence of amino acids forming the backbone of proteins
(polypeptides).
2. Secondary structure: The spatial arrangement of protein by twisting of the polypeptide chain.
3. Tertiary structure: The three dimensional structure of a functional protein.
4. Quaternary structure: Some of the proteins are composed of two or more polypeptide chains
referred to as subunits. The spatial arrangement of these subunits is known as quaternary
structure
The term protein is generally used for a polypeptide containing more than 50 amino acids

Lipids

• A lipid is any of various organic compounds that are insoluble in water. They include fats,
waxes, oils, hormones, and certain components of membranes and function as energy-storage
molecules and chemical messengers.
• A phosphor lipid/ membrane lipid is made of glycerol head and 2 fatty acid tails. A triglyceride
has Glycerol head and 3 fatty acid tails.
• Cholesterol is a waxy, fat-like substance that's found in all the cells in body.

Phospholipid – membrane lipid

Nucleic acid

• There are two types of nucleic acids, namely deoxy ribonucleic acid (DNA) and ribonucleic acid
(RNA). Primarily, nucleic acids serve as repositories and transmitters of genetic information.

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 • Nucleic acids are the polymers of nucleotides (polynucleotides) held by 3' and 5' phosphate
bridges.
• Nucleotides are composed of a nitrogenous base, a pentose sugar and a phosphate. The term
nucleoside refers to base + sugar. Thus, nucleotide is nucleoside +phosphate
• The pentose sugars are Deoxy ribose in DNA and ribose in RNA.
• DNA and RNA contain the same purines namely adenine (A) and guanine (C). Further the
pyrimidine cytosine (C) is found in both DNA and RNA. However, the nucleic acids differ with
respect to the second pyrimidine base. DNA contains thymine (T) whereas RNA contains uracil
(U).
• Difference between DNA and RNA
DNA RNA
Double stranded Single stranded
Bases are Adenine, Guanine, Thymine, Bases are Adenine, Guanine, Uracil, Cytosine.
Cytosine.
Pentose sugar is Deoxy ribose Pentose sugar is ribose
DNA stores the information RNA expresses the information
DNA is the genetic material in higher RNA is genetic material in only few viruses
organisms.
Length of DNA is quite large RNA is shorter
More stable compare to RNA Not as stable as DNA

Diversity of bonds in biomolecules

1. Ionic bond
• An ionic bond is formed by the complete transfer of some electrons from one atom to another.
• The simplest and most common example is the attraction between sodium (chemical name Na),
and chlorine (Cl), to form NaCl, sodium chloride, common table salt. The outermost level of a
sodium atom only has one electron, and can easily give it up in a chemical reaction.

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2. Covalent bond
• These bonds are stronger and much more common than are ionic bonds.
• These are formed by sharing of electrons.
• Covalent bonds form when electrons are shared between atoms and are attracted by the nuclei of
both atoms

3. Hydrogen bond
• is a special form of dipole-dipole attraction between molecules. It arises from the attractive force
between a covalently bound hydrogen atom with a very electronegative atom such as an atom of
N, O, or F.

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4. Van der Waals interactions
• intermolecular interactions that do not involve covalent bonds or ions.
• Is a temporary attractive force between atoms due to the formation of temporary dipole.

Primary Bonds- These are the covalent bonds formed as a result of electron sharing among two or
more atoms. They are formed as a result of a chemical reaction that may be reversible or irreversible.
Primary bonds are the permanent attractions that are developed among the atoms by the sharing of
electrons

1. Glycosidic bond
• It is a primary bond or a covalent bond that serves to connect carbohydrates to other groups
or molecules. The partner or combining molecule may be carbohydrate or non-carbohydrate
in nature.
• This bond is formed as a result of a reaction between the carbonyl group of a carbohydrate or
its derivate and a hydroxyl group of some other compound. The carbonyl group of
carbohydrate may be a part of an aldehydic group or a ketonic group. A molecule of water is
released in this process, making it an irreversible reaction.

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2. Peptide bond
• When the amino group of an amino acid combines with the carboxyl group of another amino
acid, a peptide bond is formed
• Dipeptide will have two amino acids and one peptide (not two) bond. Peptides containing more
than 10 amino acids (decapeptide) are referred to as polypeptides.

3. Ester bond
• An ester group is formed by dehydration of an acid and an alcohol group.
• An ester bond is formed when a molecule having the carboxylic group reacts with another
molecule having a hydroxyl group. The carboxylic group loses its hydrogen and oxygen while
the alcohol loses hydrogen of its hydroxyl group. As a result, a water molecule is released, and
the two carbons are linked via an oxygen bridge forming a -COC- linkage.

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 4. Phosphodiester bond
• It is the primary covalent bond that joins different nucleotides in a polynucleotide or nucleic
acids. It is also a type of ester bond but involves two ester linkages.
• A phosphodiester bond is a double ester linkage formed when the phosphate group at the 5’ end
of one nucleotide reacts with the free hydroxyl group at the 3’ end of another nucleotide. A
molecule of water is released, and two ester linkages are formed. In these linkages, the oxygen
bridge is used to connect a carbon atom with a phosphate group.
• Phosphodiester bonds are used to attach nucleotides in DNA and RNA.

Secondary bonds

• The secondary bonds in biological molecules are the temporary forces of attractions that are
developed when certain atoms or groups come close together. These bonds are mainly involved
in maintaining the secondary, tertiary or other higher structures of biological molecules. They are
most important in proteins and nucleic acids.

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Hydrogen bond

• Hydrogen bond results from the attractive force between hydrogen atom of one molecule and
electronegative atom such as O or N from another molecule
• In water H2O, The two hydrogen with a positive charge is connected to oxygen which is
partially negative in nature Hence Hydrogen bonding occurs between

Hydrophobic Interactions

• These are the interactions among the non-polar molecules. Such molecules cannot dissolve in
water. However, they tend to clump together away from the polar or charged molecules. This
clumping of hydrophobic molecules is called hydrophobic interaction.

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Transcription

• The process of synthesis of mRNA from DNA is known as transcription. Transcription is the
first step of gene expression.
• Transcription process in prokaryotes is very simple whereas in eukaryotes it is very complex.
• The polarity of DNA or the direction of DNA strand is indicated by the carbon numbering of the
pentose sugar.
• DNA strands are antiparallel i.e., they run in the opposite direction. One of the strands is in 3’ →
5’ direction whereas the other strand runs in 5’ → 3’ direction.
• Only one of the strands that runs in 3’ → 5’ direction can lead to the synthesis of mRNA. This
strand is known as the template strand or noncoding strand or antisense strand.

• Synthesis of RNA is catalyzed by the enzyme RNA polymerase enzyme.

• Process of transcription begins at a particular conserved sequence called promoter and continues
until it reaches terminator sequence.
• The DNA sequence occurring between promoter and terminator region is called transcription
unit.

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The transcription bubble

• Transcription bubble is created when RNA polymerase binds to the DNA sequence.
• Transcription bubble refers to the structure formed by opening of the double helix when the
RNA polymerase enzyme binds to promoter.

General outline of transcription

1. Initiation: RNA polymerase searches for the promoter region and bind to it, as a result
transcription bubble is formed. The sequence of DNA needed for RNA polymerase to bind to the
template and accomplish initiation is called the promoter.
2. Elongation: Enzyme moves along DNA and synthesizes RNA based on the nucleotide sequence
of template strand. As the enzyme moves it unwinds the DNA to expose new segment of the
template strand. Nucleotides are covalently added to the 3’ end of the growing RNA chain.
3. Termination: Process of transcription stops when termination sequence is reached. It involves the
recognition of a point after which no further bases should be added.

Transcription in Prokaryotes

• Transcription in prokaryotes is carried out by a single RNA polymerase enzyme. It is involved in

the synthesis of rRNA, tRNA and mRNA.

Initiation

• The RNA polymerase enzyme associates with DNA and travels through the DNA to search for
the promoter sequence
• Promoter is defined by specific sequences such as TATA box (TATAAT) at – 10 position and
TTGACA sequence at -35 sequence.
• Identification of the promoter is the function of sigma ((σ)) factor which is bound to RNA
polymerase enzyme. There are several different types of sigma (σ) factors which may be
involved in the identification of different types of promoters.

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Elongation

• Based on the sequence of nucleotides in the template strand the new mRNA is synthesized. The
new mRNA is complementary to the template strand and has the same sequence as the coding
strand.

• With the release of σ factor from the preinitiation complex, elongation process starts. RNA
polymerase enzyme to proceed along the DNA template (which is in 3’ to 5’ direction),
synthesizing mRNA in the 5′ to 3′ direction at a rate of approximately 40 nucleotides per second.

• RNA polymerase binds to the 3′ end of a gene (promoter) on the DNA template strand and
travels toward the 5′ end. Based on the template it continuously bonds the nucleotides together
by phosphodiester bond.

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Termination

• Once RNA polymerase has started transcription, enzyme moves along the template until it has
reached terminator sequence. At this point the enzyme stops adding nucleotides and releases the
products.
• Two types of termination can occur in prokaryotes
1) Rho dependent termination – Special proteins called rho protein binds to rho utilization
site on RNA leading to release of RNA transcript.
2) Rho independent termination – A hairpin loop is formed in the RNA transcript which
knocks off the enzyme complex.

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Transcription in Eukaryotes

• Transcription in eukaryotes is carried out by 3 different enzymes

1) RNA polymerase I – Synthesises rRNA in nucleolus.
2) RNA polymerase II – Synthesises mRNA in nucleoplasm.
3) RNA polymerase III – Synthesises tRNA in nucleoplasm.
• Our study is limited to mRNA synthesis using RNA polymerase II

Initiation

• RNA polymerase requires set of general transcription factors initiate transcription.

• TATA box (TATAAT) is the common component of eukaryotic promoter. DPE, InR are also
promoters in eukaryotes.
• Promoter is identified by the factor TFIID. TATA Binding Protein (TBP) of TFIID identifies the
promoter.
• Followed by TFIID other transcription factors bind in defined order. TFIIA is required to provide
stability. TFIIB connects the transcription factors to the RNA polymerase enzyme. TFIIE and
TFIIH are required to melt DNA and allow polymerase movement.
• In addition to these there are activators, enhancers, silencers which are involved in initiation of
transcription.

Elongation

• Transcription elongation factor b (P - TEFb) phosphorylates Pol II enzyme and helps in moving
through elongation.
• TFIIS increases the overall rate of transcription
• SPT5 is required to recruit capping enzyme.
• The mRNA as soon as is synthesised needs to be capped. Capping is the addition of modified
guanine (methylated guanine) base to the 5’ end of RNA by 5’ – 5’ linkage.
• Once the mRNA is completely synthesised tailing or polyadenylation is carried out. Poly A
polymerase adds about 200s of adenine nucleotides to the 3’ end of the mRNA.

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Termination

• Occurs by mechanisms not clearly understood.

• Once the mRNA is released from the transcription machinery, it undergoes further processing by
a process called splicing.
• The process by which introns, the noncoding regions of genes, are excised out of the primary
messenger RNA transcript, and the exons (i.e., coding regions) are joined together to generate
mature messenger RNA which serves as the template for synthesis of a specific protein.

Difference between prokaryotic and eukaryotic transcription – Self study

Translation

mRNA

• It carries the genetic message from the chromosomes (DNA) to the ribosome (site of protein
synthesis). This RNA is formed in the nucleus (or nuclear zone in prokaryotes) containing
complementary base sequence to a part of one strand of DNA (gene).
• The base sequence in the mRNA specifies the amino acid sequence in the polypeptide chains.
• In prokaryotes a single mRNA molecule codes either for one polypeptide chain, hence called
monocistronic, or it may code for more than one polypeptide, thus called polycistronic. In
eukaryotes, most of the mRNAs are monocistronic.
• mRNA runs in 5’ → 3’ direction.
• mRNA in case of prokaryotes is made of only expressive regions or exons. In eukaryotes non
expressive regions called as introns are present in between exons.
• Eukaryotic mRNA at the 5’ end has a methyl guanine cap and poly A tail at the 3’ end.

tRNA

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• They carry amino acids and bring them to ribosomes to form the polypeptide chain.
• The universally accepted 2-dimensional model of tRNA is the “clover leaf model”.
• The 5′ P end terminates usually into guanine (G), while the 3′ OH end always terminates into a
5′ CCA 3′ sequence.
• The terminal A residue is the site at which the amino acid is bound covalently.
• Nearest to the 3’ end is the T loop, followed by the variable loop whose length can vary
depending on the tRNA. Near the 5’ end of the tRNA is the D loop.
• The anticodon loop has 3 nucleotide sequences which are complementary to the codon of the
mRNA (which is why they have the name anti- codon).
• Anticodon helps in the proper identification of codon of the mRNA so that correct amino acid
may be brought based on the sequence in the mRNA.
• The enzyme aminoacyl tRNA synthetase is responsible for bonding together the amino acid and
the tRNA. Based on the anticodon sequence the enzyme attaches the respective amino acid.
• The process of attaching an amino acid to its corresponding transfer RNA (tRNA) is called
amino acid activation, also known as tRNA charging or aminoacylation.
• Aminoacyl-tRNA is tRNA to which its respective amino acid is chemically bonded (charged).
• Aminoacyl tRNA synthetase utilises ATP to form this bond.

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Ribosome

• Ribosomes are composed of two subunits, one small and one large. Prokaryotic ribosome is 70S
where the larger subunit is 50S and the smaller subunit is 30S. Eukaryotic ribosome is 80S, made
of 60S and 40S subunits.
• Ribosome is a nucleoprotein particle i.e., the ribosome is made of proteins and RNA.
• Ribosomes are capable of self- assembly and assemble just before translation.
• During translation the ribosome interacts with mRNA as well as tRNA so that based on the
sequence of mRNA, the tRNA can bring the amino acids and the aminoacids are bonded together
to make a polypeptide.
• The three tRNA sites are labeled P, A, and E. The P site, called the peptidyl site, binds to the
tRNA holding the growing polypeptide chain of amino acids. The A site (acceptor site), binds to
the aminoacyl tRNA, which holds the new amino acid to be added to the polypeptide chain. The
E site (exit site), serves as a threshold, the final transitory step before a tRNA now bereft of its
amino acid is let go by the ribosome.

• Protein Synthesis Occurs by Initiation, Elongation, and Termination.

• During translation Based on the sequences on mRNA, Amino acid is brought by tRNA and
synthesis of polypeptide occurs in presence of ribosomes.

• A specific codons codes for a specific amino acid. Different codons and amino acids coded by
them are summarized in a table.

• There are a total of 64 codons which code for 20 naturally occurring amino acids.

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 • The ribosome has three tRNA-binding sites.

• An aminoacyl-tRNA enters the A site.

• Peptidyl-tRNA is bound in the P site.

• Deacylated tRNA exits via the E site.

• An amino acid is added to the polypeptide chain by transferring the polypeptide from peptidyl-
tRNA in the P site to aminoacyl-tRNA in the A site.

Outline of translation

1. Initiation – Process of translation starts at the start codon AUG which codes for methionine.
2. Elongation – The tRNA continuously brings the amino acids based on the codons of amino acids
and peptide bond is formed between them.
3. Termination – occurs when any of the three codons UAA, UAG, UGA reaches the A site process
of translation stops.

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Prokaryotic translation

Initiation

• In prokaryotes the initiation site is the start codon. The start codon is usually 5’AUG3’,
sometimes 5’ GUG 3’ and rarely 5’ UUG 3’.
• The mRNA has ribosome binding site on the mRNA called as Shine Dalgarno sequence (5’
AGGAGG 3’) is responsible for interacting with the rRNA sequences such that AUG sequence is
directly above the P site of ribosome.

• Larger subunit of ribosome joins just before the elongation process begins.
• Initiation factors involved
1. IF1 – It blocks the P site of the ribosome
2. IF2 - With the help of GTP hydrolysis helps in attaching the initiator tRNA to the codon
at A site which is the start site
3. IF3 – It blocks the E site and also prevents the binding of the larger subunit of ribosome
before initiation step is complete and elongation can begin.
• Once the initiator tRNA is present at the start codon, the initiation factors are shed and
elongation process begins.

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Elongation

• During elongation amino acid bearing tRNAs (Aminoacyl tRNA) enter the A site. Peptide bond
is formed between the methionine in the Initiator tRNA and the amino acid attached to the
aminoacyl tRNA.
• The initiator tRNA then moves to the E site and is later released. Simultaneously the tRNA at the
A site which now contains a dipeptide move to the P site and the A site is available for new
tRNA. In order to expose new codon at the A site translocation occurs.
• Translocation is the process that advances the mRNA–tRNA moiety by one codon on the
ribosome, to allow the next codon to move into the decoding center or the A site.

• Elongation factors involved

1. EF-Tu: Brings the tRNA to the A site, ensures correct codon and anticodon match.
Process continues only if the codon =s and anticodons match.
2. EF-G: Required for translocation.

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Termination of translation

• Occurs when either of the three stop codons (UAA, UAG, UGA) come at the A site.
• Stop codons are recognised by release factors that lead to the release of polypeptide from the
tRNA along with the separation of 2 subunits of ribosome.
• Release factors involved
1. RF1 – UAG, UAA
2. RF2 – UGA, UAA
3. RF3 – Stimulate the release of RF1 and RF2

Translation in eukaryotes

Initiation

• The small subunit which is bound to the initiator tRNA binds to the 5’ end of the mRNA by
identifying the cap.
• It then scans the mRNA until it reaches the first AUG which will be considered as the start point
of translation.
• Initiation factors involved
1. eIF1, eIF5 – Function similar to IF1 and IF3 of prokaryotes
2. eTF2 – brings tRNA to A site
3. eIF4E – cap binding protein which is required for the recognition of 5’ cap
4. eIF4G – increases the level of translation
• Correct pairing of initiator tRNA leads to removal of all the initiation factors

Elongation

• Procedure same as prokaryotes

• Elongation factors involved
1. eEF1 – Required to bring tRNA to A site
2. eEF2 – For translocation

Termination

• Occurs when the stop codons are recognised by release factors

• Release factors involved
1. eRF1 – Recognises UAA, UAG, UGA
2. eRF3 – For dissociation of release factors.

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Difference between prokaryotic and eukaryotic translation

Prokaryotes Eukaryotes

Transcription and translation are coupled. Translation occurs in cytoplasm whereas

transcription occurs in nucleus.
70S ribosome is involved 80 S ribosome is involved

Cap independent initiation of translation Cap dependent initiation of translation

Shine Dalgarno sequence involved in initiation Shine Dalgarno sequence absent

Formylated methionine is the first amino acid No formylation of Amino acid

Initiation factors are IF1, IF2, IF3 Initiation factors are eIF1, eIF2, eIF4, eIF5

Elongation factors are EF – Tu, EF – Ts Elongation factors are eEF1, eEF2

Termination factors RF1, RF2, RF3 Termination factors eRF1, eRF3

Post translational modification of polypeptide Post translational modification occurs in the

occurs in the cytoplasm endoplasmic reticulum

Stem cells and its applications – Refer ppt

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