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Dna Damage and Repair - Lecture notes, lectures 1 - 3

Molecular and Cellular Biochemistry: Molecular Cell Biology (University of Southampton)

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DNA Damage and Repair

Types of DNA Damage:
1. Replication errors
Repair Pathway followed: MMR
2. Base tautomers
♥ Base tautomerism may result in either Transition or Transversion
♥ Transition: GC - AT; AT - GC (Order of Purines and Pyrimidine is conserved)
♥ Transversion: GC - TA or CG; AT - CG or TA (Order of Purines and Pyrimidines is reversed)

Cytosine Deamination
♥ Cytosine in DNA spontaneously deaminates to form Uracil.
♥ Deamination of Cytosine is mutagenic because Uracil pairs with Adenine and so one of the
daughter strands will contain a U-A base pair rather than the original C-G base pair.

3. Covalent modification
 Oxidative Damage
Oxidative damage to Guanine
♥ Mutagens include reactive oxygen species, such as hydroxyl radical.
♥ Hydroxyl radical reacts Guanine to form 8-Oxoguanine.
♥ 8-Oxoguanine is mutagenetic because it often pairs with Adenine rather than Cytosine in DNA
replication.
♥ Repair Pathway followed: BER

 UV-damage (Cytosine)
 Ultraviolet component of sunlight is a ubiquitous DNA-damaging agent
 Major effect is to covalently link adjacent pyrimidine residues along a DNA strand.
 Such a pyrimidine dimer cannot fit into a double helix, and so replication and gene expression are
blocked until the lesion is removed.
 Example of an Intrastrand Cross-link is a Thymine Dimer.
 Both the participating bases are in the same strand of the double helix.
 Cross-links between bases on opposite strands can also be introduced by various agents.
 Psoralen (Compounds produced by a Chinese herb), forms such Interstrand Cross-Links.
Interstrand cross-links disrupt replication since they prevent strand separation.
♥ Repair Pathway followed: Direct Reversal, NER

 Chemical Damage (including alkylation)

Deamination of Adenine
♥ Adenine can also be deaminated by Nitrous Acid (HNO2) to form Hypoxanthine.
♥ The process is mutagenic because Hypoxanthine pairs with Cytosine rather than Thymine.
♥ Repair Pathway followed: BER

Alkylation
♥ Nucleotide bases are subject to Alkylation.
♥ Electrophilic centers can be attacked by nucleophiles such as N-7 of Guanine and Adenine to
form alkylated adducts.

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♥ Some compounds are converted into highly active electrophiles through the action of enzymes
that normally play a role in detoxification.
♥ Example: Aflatoxin B1 is a compound that is produced by mould on peanuts and other foods.
♥ Cytochrome p450 enzyme converts this compound into a highly reactive Epoxide which then
reacts with N-7 atom of Guanosine to form a mutagenic adduct that frequently leads to a G-C to
T-A transversion.
♥ Repair Pathway followed: Direct Reversal

 Electromagnetic radiation damage

♥ High-energy electromagnetic radiations such as X-rays can damage DNA by producing high
concentrations of reactive species in solution,
♥ X-ray exposure can induce DNA damages likes single- and double-stranded breaks in the DNA.
♥ Repair Pathway followed: Non-homologous end joining

4. Non-covalent Interactions

DNA Repair Mechanisms

♥ Many systems repair DNA by using sequence information from the uncompromised strand.
♥ Such single-strand replication systems follow a similar mechanistic outline:
A. Recognize the offending base(s)
B. Removal of the offending base(s)
C. Repair the resulting gap with a DNA Polymerase and DNA Ligase.

Types of Repair Mechanisms:

1. Direct Repair
♥ Example: Photochemical cleavage of Pyrimidine Dimers.
♥ All cells contain a Photoreactivating Enzyme called DNA Photolyase.
♥ The E.Coli enzyme contains bound N^5,N^10-methenyltetrahydrofolate and Flavin Adenine
Dinucleotide (FAD) cofactors.
♥ The enzyme binds to the distorted region of DNA.
♥ Using light energy - specifically, absorption of a photon by N^5,N^10-
methenyltertahydrofolate coenzyme leads to the formation of an excited state that cleaves the
dimer into its component bases.

2. Base Excision Repair

♥ Involves the excision of modified bases such as 3-methyladenine by the E.Coli enzyme, AlkA.
♥ Binding of the enzyme to damaged DNA, flips the affect base out of the DNA double helix and
into the active site of the enzyme.
♥ Enzyme then acts as a Glycosylase, cleaving the Glycosidic Bond to release the damaged base.
♥ At this stage, the DNA backbone is intact but it missing a base - This hole is known as the AP
Site because it is either Apurinic (Devoid of A or G) or Apyrimidinic (Devoid of C or T).
♥ An AP Endonuclease recognizes this defect and nicks the backbone adjacent to the missing
base.

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♥ Deoxyribose Phosphodiesterase excises the residual deoxyribose phosphate unit, and DNA
Polymerase I inserts an undamaged nucleotide as dictated by the base on the undamaged
complementary strand.
♥ The repaired strand is finally sealed by DNA Ligase.

3. Nucleotide Excision Repair (NER)

♥ Utilized best for the excision of Pyrimidine Dimers.
♥ 3 enzymatic activities are essential for this repair process.
♥ First, an enzyme consisting of the proteins encoded by the UvrABC genes detect the distortion
produced by the DNA damage.
♥ UvrABC enzyme then cuts the damaged DNA strand at 2 sites, 8 nucleotides away from the
damaged site on the 5' side and 4 nucleotides away on the 3' side.
♥ The 12-residue oligonucleotide excised by this Excinuclease then diffuses away.
♥ DNA Polymerase I enters the cap to carry out the repair synthesis.
♥ 3' end of the nicked strand is the primer, and the intact complementary strand is the template.
♥ Finally, 3' end of the newly synthesised stretch of DNA and the original part of the DNA chain
are joined by DNA ligase.

4. Mismatch Repair
♥ Consists of at least 2 proteins, 1 for detecting the mismatch and the other for recruiting an
endonuclease that cleaves the newly synthesised DNA strand close to the lesion to facilitate
repair.
♥ In E.Coli, these proteins are called MutS and MutL and the endonuclease is MutH.
MutS recognizes and binds to the mismatch

 This mutation is prevented by a repair system that recognizes Uracil foreign to DNA.
 The repair enzyme, Uracil-N-Glycosylase (Type of Uracil DNA Glycosylase is homologous to AlkA.
 Enzyme hydrolyses the glycosidic bond between Uracil and deoxyribose moieties but does not
attack thymine-containing nucleotides.
 AP site generated is repaired to reinsert Cytosine.
 Thus, the methyl group on Thymine is a tag that distinguishes Thymine from Deaminated Cytosine.

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1. Initiation: Before DNA Replication begins, parental strands must be separated and stabilised
transiently in the single-stranded state. After this stage, synthesis of daughter strands can be
initiated at the replication fork.
 Initiation of replication takes place at a fixed sequence (OriC) from which the replication forks
move bidirectionally until they reach the terminal sequence terC
 DnaA protein specific for initiation of replication
2. Elongation: Complex of proteins called Replisome involved in this stage. Does not exist as an
independent unit but instead, forms a protein complex with the particular structure that the DNA
takes up at the replication fork. As replisome moves along parental strand, DNA helix unwinds and
daughter strands are synthesised.
3. Termination: Termination required following DNA Replication and separation of two identical
chromosomes produced required which requires manipulation of higher order DNA structure.

DNA Replication first requires a Helicase and Single-Strand Binding Protein (SSBP)
 Helicase is an enzyme that separates (melts) the strands of DNA, using hydrolysis of ATP to
provide the necessary energy.
 Helicase has one conformation that binds to duplex DNA and another conformation that binds to
single stranded DNA.
 Alternation between the two conformations is what drives the motor to melt DNA and requires
ATP hydrolysis - 1 ATP molecule is hydrolysed for each base pair that is unwound.
 Helicase cannot initiated unwinding of duplex DNA, it can only initiate unwinding at a single-
stranded region adjacent to a duplex.

 SSBP binds to the single-stranded DNA, protecting it and preventing it fro reforming the duplex
state. SSBP binds in a cooperative manner, binding of additional monomers to the existing
complex is enhanced.
E.Coli SSB - Tetramer of 74 kD, Eukaryotic SSB - Trimer
 Unwinding by Helicase generates 2 strands which are then bound by SSB.
 Significance of cooperative binding is such that binding of one protein molecule makes it much
easier for another to bind.
 Thus, once binding reaction has started on a particular DNA molecule, it is rapidly extended until
all of the single-stranded DNA is covered with SSB protein.
 SSB is not a DNA unwinding protein, its main function is to stabilize DNA that is already in the
single-stranded condition.
 SSB binds as the replication fork advances, keeping the two parental strands separate so that they
are in the appropriate condition to act as templates.

Following unwinding by Helicase and stabilization by SSBP, a Primer is required to start

DNA Synthesis
 DNA Polymerases cannot initiate synthesis of a chain of DNA de novo, but can only elongate a
chain.
 Synthesis of a new strand can only occur from a preexisting 3'-OH end and the template strand
must be converted to a single-stranded condition
 3'-OH end is called a Primer
 Sequence of RNA is synthesised on the template so that the free 3'-OH end of the RNA chain is
extended by DNA Polymerase; OR
 Preformed RNA (tRNA) pairs with the template, allowing its 3'-OH end to be used to prime DNA
synthesis.

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 Primer is required to provide 3'-OH end to start off the DNA chains on both leading and lagging
strands.
 Leading Strand only requires one such initiation event, whereas,
 Lagging Strand requires a series of initiation events due to each Okazaki fragment requiring its
own star de novo.
 Each Okazaki fragment starts with a primer sequence of RNA approx. 10 base pairs long that
provides the 3'-OH end for extension by DNA Polymerase

Primase (dnaG) is required to catalyze the actual priming reaction

 Primase is an RNA Polymerase that is used only under specific circumstances i.e. to synthesize
short stretches of RNA that are used as primers for DNA synthesis.

A Helicase is required to generate single strands, a Single-Strand Binding Protein is required

to maintain the single-stranded state and the Primase synthesizes the RNA primer

DNA Polymerases
i. DNA Polymerase I involved in repair of damaged DNA and in a subsidiary role of semiconservative
replication
ii. DNA Polymerase II required to restart progress of replication fork when it is stopped by DNA
damage
iii. DNA Polymerase III responsible for de novo synthesis of new strands of DNA. Multisubunit
protein.

iv. DNA Polymerase I: -

 Along with the ability to synthesize DNA in 5'-3' direction, replicases also have 3'-5' nuclease
activity.
 3'-5' Nuclease activity required to excise bases that have been added to the DNA incorrectly -
Serves as a 'proofreading' mechanism.
 Single polypeptide of 103 kD, chain can be cleaved into two parts by proteolytic treatment.
 Larger cleavage product - Klenow fragment (68 kD) - possesses polymerase and proofreading
activities.
 Smaller cleavage product (35 kD) possesses 5'-3' exonucleolytic activity. Excises small groups of
nucleotides (approx. 10 bases at a time). Coordinated with proofreading activity.

A. Nick Translation Function of DNA Polymerase I: -

 Unique ability of the enzyme to start replication at a nick in the DNA.
 At a point where a phosphodiester bond has been broken in a double stranded DNA, enzyme
extends the 3'-OH end.
 As the new segment of DNA is synthesized, it displaces the existing homologous strand in the
duplex.
 Displaced strand is degraded by the 5'-3' exonucleolytic activity of the enzyme.
 Properties of DNA remain unaltered, except that a segment of the DNA has been replaced by a
newly synthesized segment and the nick has been moved further along the duplex.
 Coupled 5'-3' synthetic/3'-5' exonucleolytic activity is used most extensively to fill in short single-
stranded regions in double-stranded DNA.
 Such regions arise during lagging strand DNA replication.

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DNA Polymerase Common Structure:

 Enzyme structure divided into independent domains described using the analogy of a human right
hand.
 DNA binds in a large cleft of the 3 domains.
 "Palm" of the enzyme contains conserved sequence motifs which provide the catalytic active site
 "Fingers" allow for correct positioning of the parent template at the active site.
 "Thumb" binds the DNA as it exists the enzyme, and is important in processivity.
 Exonuclease activity of the enzyme exists on an independent domain with its own catalytic site.

Leading and Lagging Strands

 Problem posed by structure of DNA: Antiparallel structure of the two strands of duplex DNA pose
a problem for replication and Coiling of the two strands around each other
 As the replication fork advances, daughter strands must be synthesised on both of the exposed
parental single strands.
 Fork template strand moves in 5'-3' direction on one strand and 3'-5 direction on the other strand.
 Since overall direction of synthesis must remain 5'-3', the problem is solved by synthesizing the
new strand on the 5' to 3' template in short fragments/each synthesized in the backwards
direction.

 Leading Strand (Forward Strand): DNA synthesis can proceed continuously from 5'-3' direction as
the parental DNA duplex is unwound.
 Each new DNA strand, leading and lagging strand is synthesized by an individual catalytic unit.
 One enzyme unit is moving in the same direction as the unwinding point of the replication fork
and synthesizing the leading strand continuously
 Lagging Strand (Backward Strand): Stretch of single-stranded parental DNA must be exposed, the
daughter strand is then synthesised in the reverse direction relative to fork movement. A series of
fragments (Okazaki fragments-Found in prokaryotes and eukaryotes) are synthesised in the 5'-3'
direction and are then joined together to create an intact lagging strand - Semi discontinuous
Replication.
 The other enzyme unit is moving backward relative to the DNA.
 When synthesis of one Okazaki fragment is completed, synthesis of the next Okazaki fragment is
required to start at a new location approximately in the vicinity of the growing point for the
leading strand.
 This requires DNA Polymerase III to disengage from the template and reconnect to the template
at a new location at a primer to start a new Okazaki fragment.

 As the Replisome (DNA Pol III) moves along DNA, unwinding the parental strands, one enzyme unit
elongates the leading strand.
 Periodically, the Primosome activity initiates an Okazaki fragment on the lagging strand and the
other enzyme unit must then move in the reverse direction to synthesize DNA.

DNA Polymerase III (Replisome) Holoenzyme and its Sub complexes

 Three-polymerase core structure i.e. 2 Pol III catalytic cores responsible for synthesis of lagging
strand and 1 Pol III catalytic core responsible for leading strand.
 Holoenzyme is a complex of 900 kD that contains 10 different proteins organized into 4 types of
subcomplex:

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 Each catalytic core consists of an Alpha subunit which carries out DNA Polymerase activity, E
subunit that carries out 3'-5' exonuclease activity and the Theta subunit which stimulates
exonuclease
 There are 2 copies of the dimerizing subunit (Pi) which link the 2 catalytic cores together and
maintain dimeric structure.
 There are 2 copies of the Beta clamp which is responsible for holding catalytic cores onto their
template strands. Each clamp consists of a homodimer of B subunits, B2 ring, which bind around
the DNA and ensures processivity.
 B is strongly bound to DNA but provides the sliding clamp that allows the holoenzyme to slide
along the duplex molecule.
 Ring shaped dimer is formed is formed around DNA and the space between the protein ring and
DNA is fill by water.
 Structure explains high processivity - The enzyme can transiently dissociate but does not fall off
and diffuse away.
 Alpha helices on the inside of the Beta clamp have some positive charges that may interact with
DNA via the water molecules.
Loading of Beta Clamp: The clamp is a circle of subunits surrounding the DNA, its assembly or
removal requires the use of an energy dependent process by the clamp loader.
 The Gamma complex is a group of 7 proteins, encoded by 5 genes that comprise the clamp loader
- Clamp loader cleaves ATP to load the clamp on DNA.
 Gamma clamp loader is a pentameric circular structure that binds an open form of the B2 ring
preparatory to loading it onto DNA.
 Binding of Gamma to the ring, destabilizes it and opens it, facilitated by ATP.
 While one polymerase subunit of DNA Polymerase synthesizes the leading strand continuously,
the other cyclically initiates and terminates the Okazaki fragments of the lagging strand.
 Replication fork created by Helicase which forms a hexameric ring - Translocates in the 5'-3'
direction on the template for the lagging strand.
 Helicase is connected to the two DNA Polymerase catalytic units each of which is associated with a
sliding clamp.

Mechanism and Structure of DNA Polymerase III

 A catalytic core is associated with each template strand
 Holoenzyme moves continuously along the template for the leading strand
 Template for the lagging strand is 'pulled through' thus creating a loop in the DNA.
 DnaB contacts the Pi subunits of the clamp loader.
 A direct connection is established between the helicase-primase complex and the catalytic cores.
There are two effects of the links: -
i. Increase in the speed of DNA synthesis by increasing the rate of movement by DNA Polymerase
tenfold.
ii. Prevent the leading strand polymerase from falling off, i.e. to increase processivity.
 Synthesis of leading strand creates a single stranded DNA loop which proves the template for
lagging strand synthesis.

On completion of Okazaki fragment

 All components of the replication apparatus function processively except Primase and B2 clamp.
 B2 clamp must be cracked open by the Gamma clamp loader when the synthesis of each fragment
is completed.

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 Clamp loader causes B2 clamp to alter its conformation to an unstable configuration, which then
springs open.
 New B2 clamp is recruited by the clamp loader to initiate the next Okazaki fragment.
 Lagging strand polymerase transfers from one B2 clamp to the next in each cycle, without
dissociating from the replicating complex.

Okazaki Fragments are linked by DNA Ligase

 Process involves: -
A. Synthesis of RNA Primer
B. Extension with DNA
C. Removal of RNA primer
D. Replacement by a stretch of DNA
E. Covalent linking of adjacent Okazaki fragments.

 In mammalian systems, Okazaki fragments are connected in a 2 step process:

1. Synthesis of Okazaki fragment displaces the RNA Primer of the preceding fragment in the form of
a flap.
 Primer is cleaved by enzyme FEN1 (f lap endonuclease 1)

2. Once the RNA has been removed and replaced, the adjacent Okazaki fragments must be linked
together.
 3'-OH end of one fragment is adjacent to the 5'-phosphate end of the previous fragment.
 Enzyme DNA Ligase make a bond by using a complex with AMP.
 AMP of the enzyme complex becomes attached to the 5' phosphate of the nick and then a
phosphodiester bond is formed with the 3'-Oh terminus of the nick,
 Releasing the enzyme and the AMP.

Topoisomerases
 Isomerase enzymes that regulate the overwinding or underwinding of DNA and act on the
topology of DNA
 During DNA Replication and Transcription, DNA becomes overwound ahead of a replication fork ,
this tension would eventually stop the ability of the enzymes involved in these processes to
continue down the DNA strand.
 They relieve the super helical stress that is produced in front of and behind the replication fork.
 They also separate the two daughter DNA molecules after the cycle of replication is complete.
 In order to prevent these topological problems caused by double helix, topoisomerases bind to
either Single-Stranded DNA or Double-Stranded DNA and cut off the phosphate backbone of the
DNA.
 An enzyme-DNA complex is formed linked the nicked phosphate and tyrosine in the active site of
the protein.
 Complex then swivels rotating one strand around the other and the nick is then released.
 Intermediate breaks allow for the DNA to be untangled or unwound and at the end of these
processes, the DNA backbone is resealed again.
 Topoisomerase I and III cuts 1 strand and unwinds the DNA - Decreases L by 1.
 Topoisomerase II and IV cuts both strands of a duplex and passes on strand through the other -
Decreases L by 2.

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Ribosome Structure and Translation

Genetic Code
♥ Genetic code uses 3 RNA bases to specify 1 amino acid and the genetic code is therefore, a
Triplet Code.
♥ Each of the 20 amino acids is specified by 1 or more triplets constituted from the four mRNA
bases A, U, G and C.
♥ Coding regions is a continuous sequence of non-overlapping triplets running from the 5'-end to
the 3'-end in the mRNA.
♥ Triplets in mRNA are Codons.
♥ mRNAs also contain non-coding or untranslated regions at their 5' and 3' ends.

Translation is the process by which the genetic message, encoded originally in the sequence
of the four bases A,T, C and G in DNA, is expressed in the form of an amino acid sequence in a
protein using the 20 amino acids.

tRNA - The Adaptor Molecule

♥ Transfer RNA serves as the adaptor molecule that binds to a specific codon and brings with it an
amino acid for incorporation into the polypeptide chain.
♥ tRNA consists of an amino-acid attachment site and a template- recognition site.
The carboxyl group of this amino acid is esterified to the 3' or 2' hydroxyl group of the ribose unit
at the 3' end of the tRNA chain in the form of an aminoacyl tRNA ester.
♥ Its subsequent hydrolysis helps drive peptide bond formation.
♥ Joining of an amino acid to a tRNA molecule to form an aminoacyl-tRNA is catalysed by a
specific enzyme called aminoacyl-tRNA synthetase also called activating enzymes.
♥ This esterification reaction is driven by ATP cleavage.
♥ Template-recognition site on tRNA is a sequence of 3 bases called an Anticodon.
♥ The anticodon on tRNA recognises a complementary sequence of 3 bases called a Codon on
mRNA.

Amino acid -------------------Amino acyl tRNA -------- Polypeptide one amino acid longer
Amino acyl Ribosome
tRNA synthetase peptidyl
transferase
♥ Features of tRNA: -
 Each is a single chain containing between 73 and 93 ribonucleotides
 The molecule is L-shaped with highly conserved tertiary structures with much internal base
pairing.
 tRNA possess an Anticodon that recognises the mRNA codon by anti-parallel base pairing.
 tRNAs contain numerous post-transcriptionally modified bases
 3-terminus of every tRNA ends in CCA with each amino acid linked as an ester to the 3'-OH of the
last amino acid.

Amino Acids are first activated by Acetylation

♥ Activation reaction is catalysed by specific aminoacyl tRNA synthetases
1. First step is the formation of an aminoacyl-adenylate from an amino acid and ATP

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Amino Acid + ATP = Aminoacyl-AMP + PPi

♥ The activated species is a mixed anhydride in which the carboxyl group of the amino acid is
linked to the phosphoryl group of AMP; also known as aminoacyl-AMP.

2. Next step, is transfer of the aminoacyl group of aminoacyl-AMP to a particular tRNA molecule to
form an aminoacyl-tRNA

Aminoacyl-AMP + tRNA = Aminoacyl tRNA + AMP

♥ Activation and transfer steps are catalysed by the same aminoacyl tRNA synthetase
♥ Aminoacyl-AMP does not dissociate from the synthetase, it remains tightly bound to the active
site of the enzyme by non covalent interactions.
♥ Each time, the equivalent of 2 ATP molecules is consumed in the synthesis of aminoacyl-tRNA.
♥ One of them is consumed in forming the ester linkage of aminoacyl-tRNA, whereas, the other is
consumed in driving the reaction forward.

Increased fidelity of protein synthesis by proofreading function of aminoacyl-tRNA

synthetase
♥ Each aminoacyl tRNA synthetase takes advantage of the properties of its amino acid substrate.
♥ Certain aminoacyl-tRNA synthetases for instance threonyl-tRNA synthetase contains an
additional function site that hydrolyses the incorrectly paired tRNA. In this case, Ser-tRNA but not
Thr-tRNA
♥ The editing site is more than 20 A away from the activation site.
♥ This editing site readily accepts and cleaves Ser-tRNA but not Thr-tRNA.
♥ These complementary pairs of sites function as a double sieve to ensure very high fidelity.
♥ Acylation site rejects amino acids that are large than the correct one because there is
insufficient room for them, whereas the hydrolytic site cleaves activated species that are smaller
than the correct one.
♥ The flexible CCA arm of an aminoacyl-tRNA can move the amino acid between the activation
and editing site and so the aminoacyl-tRNA can be edited without dissociating from the
synthetase.
♥ Thus, if an amino acid is linked to the wrong tRNA, then the aminoacyl tRNA still recognizes the
anticodon and inserts the wrong amino acid.

Two Classes of Aminoacyl-tRNA Synthetases: -

 Class I aminoacyl-tRNA synthetases
♥ They acylate 2'-Hydroxyl group of the terminal ribose of tRNA
♥ Most are monomeric
 Class II aminoacyl-tRNA synthetases
♥ They acylate 3'-Hydroxyl group of the terminal ribose of tRNA.
♥ Most are dimeric

 The two classes bind ATP in different conformations.

♥ Experimental evidence that anticodon recognizes the mRNA codon and not the amino acid: -

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 Cysteine-tRNAcys prepared initially which is then converted to Alanine-tRNAcys by the reduction of

-CH2-SH group into CH3 (Alanine) group in Raney Nickel
 Modified Alanine-tRNA then used in synthesis of haemoglobin in vitro.
 As a result, Alanine incorporated into the haemoglobin instead of Cysteine.

Key roles of tRNA (Summary)

♥ To activate the amino acid as a tRNA ester
♥ To act with the correct synthetase to ensure that the correct amino acid is linked to its tRNA.
♥ To bring the correct amino acid to the ribosome
♥ To base pair its anticodon triplet with the correct codon of mRNA - Decoding Role
♥ To facilitate peptide bond formation of the growing chain and the next amino acid.

Wobble in Base-Pairing
♥ Some tRNA molecules recognize more than one codon because of wobble in base-pairing.
♥ For instance, yeast alanyl-tRNA binds to 3 codons: GCU, GCA and GCC.
♥ Pattern of degeneracy of the genetic code indicates that recognition of the third codon may be
less discriminating than the recognition of the other two.
♥ The wobble hypothesis allowed for pairings at the third base of the codon according to the
following:
First base of Anticodon Third base of Codon
C G
A U
U A or G
G U or C
I U, C or A

♥ Two generalizations made concerning the anticodon-codon interaction:

 First two bases of a codon pair in the standard way. Hence, codons that differ in either of their
first two bases must be recognized by different tRNAs.
 First base of an anticodon determines whether a particular tRNA will read 1, 2 or 3 codons.
Thus, part of the degeneracy in the genetic code arises from imprecision (wobble) in the pairing
of the third base of the codon with the first base of the anticodon.

Protein Synthesis
♥ Ribosomes are the molecular machines that coordinate the interplay of charged tRNAs, mRNA
and proteins that lead to protein synthesis.
♥ Bacterial Ribosome can be dissociated into: -
 A large subunit (50S) - Contains 34 different proteins (L1 - L31) and 2 RNA molecules (23S
composed of 6 domains and 5S)
 A small subunit (30S) - Contains 21 different proteins (S1 - S19) and a 16s RNA molecule

♥ Eukaryotic Ribosome can be dissociated into: -

 A large subunit (60S) - Contains more than 49 proteins and 5S,28S and 5.8S RNA molecules.
 A small subunit (40S) - Contains more than 33 proteins and an 18S RNA molecule.

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Three binding sites that bridge the 30S and 50S subunits (Evidence of Ribosome being a
Ribozyme (3))
♥ The mRNA fragment being translated at a given moment is bound within the 30S subunit
♥ Each of the tRNA molecules is in contact with both the 30S and 50S subunit.
♥ At the 30S end, 2 of the 3 tRNA molecules are bound to the mRNA through an anticodon-codon
base pairs.
♥ These binding sites are called A site (for Aminoacyl) and P site (for Peptidyl)
♥ Third tRNA molecule is bound to an adjacent site called the E site (for Exit of deacylated tRNA).
♥ The other end of the tRNA molecule the end without the codon interacts with the 50S subunit.
♥ To create a peptide bond, NH of an incoming amino acid which is attached to tRNA is required
to attack CO of the growing polypeptide which is still attached to tRNA, ending up in an extended
polypeptide.
♥ The ribosome is a ribozyme i.e. an RNA molecule that is capable of acting as an enzyme.

Initiation of Protein Synthesis

♥ Protein synthesis in Bacteria starts with the modified amino acid N-formylmethionine (fMet).
♥ A special tRNA brings formylmethionine to the ribosome to initiate protein synthesis (tRNAf)
♥ The tRNA that inserts methionine in internal positions is (tRNAm)
♥ Methionine is linked to these two kinds of tRNAs by the same aminoacyl-tRNA synthetase.
♥ In order to bring mRNA and formylmethionyl-tRNAf, 3 initiation factors (IF1, IF2 and IF3) are
essential.
♥ 30S ribosomal unit first forms a complex with IF1 and IF3.
♥ IF1 binds near the A site and direct fMet-tRNAf to the P site.
♥ IF2, a member of G protein family, binds GTP and the conformational change enables IF2 to
associate with fMet-tRNAf.
♥ IF2-GTP-initiator-tRNA complex binds with mRNA and the 30S subunit to form the 30S initiation
complex.
♥ IF2 stimulates the association of 50S subunit to the complex.
♥ GTP bound to IF2 is hydrolysed, releasing IF2.
♥ Result: A 70S initiation complex. Formation of this complex is the rate limiting step in protein
synthesis.
♥ Once 70S initiation complex has been formed ribosome is ready for elongation phase of
protein synthesis.
♥ fMet-tRNAf molecule occupies the P site on the ribosome, so that its anticodon pairs with the
initiating codon on mRNA.
♥ The other 2 sites, A and E are empty.
♥ Bacterial translation initiation comprises of a Shine-Dalgarno sequence that base pairs to the 3'
end of the 16S rRNA.
♥ This places the start codon AUG at the ribosome P site.

Elongation of Protein Synthesis

♥ At this point, f-Met-tRNAf occupies the P site and the A site is vacant.
♥ The appropriate aminoacyl-tRNA is delivered to the A site in association with a 43-kd protein
called Elongation Factor Tu (EF-Tu) a member of the G protein family.

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♥ EF-Tu binds aminoacyl-tRNA in its GTP form and releases it to the ribosome in its GDP form.
♥ EF-Tu is reset to its GTP form by a 2nd elongation factor, Elongation Factor Ts (EF-Ts)
♥ EF-Ts induces the dissociation of GDP and peptide bond formation occurs.

Peptide-Bond Synthesis
♥ Thermodynamically stable reaction catalysed by a site on the 23S rRNA of the 50S subunit called
the Peptidyl Transferase Center with Peptidyl Transferase in Domain V
♥ This catalytic center allows the peptide to leave the ribosome
♥ Amino group of the aminoacyl-tRNA in the A site is positioned to attack carbonyl group of the
ester linkage of the peptidyl-tRNA to form a tetrahedral intermediate.
♥ This intermediate collapses to form the peptide bond and the deacylated tRNA.

(aa)n- tRNA + aa-tRNA' = (aa)n+1-tRNA' + tRNA

Peptidyl tRNA Incoming Extended peptidyl Deacylated tRNA
aminoacyl tRNA tRNA
Evidence of Ribosome being a Ribozyme (1)
♥ The Peptidyl Transferase Center, includes bases that promote this reaction by helping to form
an NH2 group on the A site aminoacyl tRNA and by helping to stabilize the tetrahedral
intermediate. There are no ribosomal proteins the PTC and ribosomes that are largely depleted of
proteins still have peptidyl-transferase activity (Evidence Ribosome being a Ribozyme (2))

Mechanism of Elongation
♥ Peptide chain is now attached to the tRNA whose anticodon is in the A site on the 30S subunit.
♥ Protein synthesis cannot continue without translocation of mRNA and tRNA within the
ribosomes.
♥ mRNA must move by a distance of 3 nucleotides so that the next codon is positioned in the A
site for interaction wit the aminoacyl-tRNA.
♥ At the same time, the deacylated tRNA moves out of the P site into the E site on the 30S subunit
and the peptidyl-tRNA moves out of the A site into the P site.
♥ Movement of peptidyl-tRNA into the P site shifts the mRNA by one codon exposing the next
codon to be translated in the A site.
♥ Translocation is enhanced by Elongation Factor G (EF-G, or known as Translocase)
♥ EF-G binds near the A site on 50S subunit of the ribosome in the GTP form.
♥ Binding of EG-F to the ribosome stimulates GTPase activity of EF-G.
♥ On GTP hydrolysis, EF-G undergoes a conformational change that displaces the peptidyl-tRNA in
the A site to the P site which carries the mRNA and deacylated tRNA with it.
♥ Dissociation of EF-G leaves the ribosome ready to accept the next aminoacyl-tRNA into the A
site.

Termination of Protein Synthesis

♥ Stop codons UAA, UGA or UAG are recognized by proteins called Release Factors (RFs)
♥ RF1 recognizes UAA or UAG
♥ RF2 recognizes UAA or UGA
♥ RF3 another GTPase mediates interactions between RF1 or RF2 and the ribosome.

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♥ When bound to the ribosome, the proteins unfold to bridge the gap between the stop codon on
the mRNA and the peptidyl transferase center on the 50S subunit.
♥ The RF interacts with peptidyl transferase center with a loop containing a highly conserved
Glycine-Glycine-Glutamine (GGQ) sequence, with a modified Glutamine.
♥ This modified Glutamine is crucial in promoting a water molecules attack on the ester linkage
between the tRNA and the polypeptide chain, freeing the polypeptide chain.
♥ The detached polypeptide leaves the ribosome.
♥ tRNA and mRNA remain briefly attached to the 70S ribosome until the entire complex is
dissociated through the hydrolysis of GTP in response to the binding of EF-G and another factor,
called the Ribosome Release Factor (RRF)

Initiation in Eukaryotes
♥ Initiating amino acid is Methionine rather than N-formylmethionine as in prokaryotes.
♥ Special tRNA participates in initiation. This aminoacyl-tRNA is called Met-tRNAi (subscript I
standing for Initiation).
♥ Initiating codon in eukaryotes is also AUG.
♥ AUG nearest the 5' end of mRNA is usually selected as a start site as opposed to specific purine-
rich sequences on the 5' side to distinguish initiator AUG from internal ones.
♥ 40S ribosome with a bound Met-tRNAi attaches to the cap at the 5' end of the eukaryotic
mRNA and searches for an AUG codon by moving step-by-step in the 3' direction.
♥ Scanning process is performed by Helicases that move along the mRNA by means of ATP
Hydrolysis.
♥ Pairing of the anticodon of Met-tRNAi with the AUG codon of mRNA signals that the target has
been found.
♥ Eukaryotic mRNA has only one start site and hence is the template for only a single protein. In
contrast, prokaryotic mRNA can have multiple start sites and it can serve as a template for the
synthesis of several proteins.
♥ Once Met-tRNAi reaches the first AUG on the mRNA, the 60S subunit is then added to form the
80S initiation complex.
♥ eIF-4E is a eukaryotic initiation factor that binds directly to 7-methylguanosine cap whereas,
eIF-2 in association of GTP, delivers the Met-tRNAi to the ribosome.
♥ eIF-4E protein that binds to the mRNA cap structure also binds to the poly(A)-tail though 2
protein intermediates.
♥ eIF-4E bound to the cap then binds to the eIF-4G protein which in turn binds to a protein
associated with the poly(A)-tail, the poly(A)-binding protein (PAPBI)
♥ Cap and tail are brought together to form a circular mRNA.

Regulation of Protein Synthesis in Eukaryotes

♥ Regulation depends on phosphorylation of Initiation Factor eIF2, which is a part of the Met-
tRNAi complex.
♥ When large numbers of eIF2 are phosphorylated, protein synthesis is inhibited.
♥ Regulator 4EBP, may bind to the Initiation Factor eIF4E found on the 5' cap on mRNA stopping
protein synthesis.

Elongation and Termination in Eukaryotes

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♥ Elongation Factors EF1Alpha and EF1BetaGamma are the counterparts of the prokaryotic EF-Tu
and EF-Ts.
♥ GTP form of EF1Alpha delivers aminoacyl-tRNA to the A site of the ribosome and
EF1BetaGamma catalyzes the exchange of GTP for bound GDP.
♥ Eukaryotic EF2 mediates GTP driven translocation.
♥ Termination is carried out by single release factor eRF1 compared with 2 in prokaryotes.
♥ eIF-3, like its prokaryotic counterpart IF3, prevents the reassociation of ribosomal subunits in
the absence of an initiation complex.

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