1.

IF1: Promotes dissociation of ribosomal subunits; stabilizes IF3 binding to 30S;

prevents premature association of 50S.
IF2: GTP-binding protein; recruits and stabilizes fMet-tRNA binding to the 30S
initiation complex; hydrolyzes GTP to release itself after 50S joins
IF3: Binds to 30S subunit; prevents premature reassociation with 50S; helps position
mRNA correctly by interacting with Shine-Dalgarno sequence.
30S Ribosomal subunit: Provides the platform for initiation; binds mRNA and fMet-
tRNA; ensures correct codon-anticodon pairing at the start site.
fMet: Specialized initiator amino acid; carried by initiator tRNA (fMet-tRNA); always
the first incorporated residue in bacterial proteins (often removed later during
maturation).
GTP: GTP hydrolysis triggers a conformational change in IF2. This change causes
IF2 (and other initiation factors like IF1 and IF3) to dissociate from the ribosome,
leaving behind the fully assembled 70S initiation complex ready for elongation.
2. Shine Dalgarno sequence: It is a ribosomal binding site found in bacterial and
archaeal mRNA. Typically located ~8 nucleotides upstream of the start codon
(AUG). The consensus sequence is AGGAGGU, though variations exist depending
on the organism.
Role of SD sequence:
 Ribosome Recruitment: The Shine-Dalgarno sequence base-pairs with a
complementary region at the 3′ end of the 16S rRNA in the 30S ribosomal subunit.
 Alignment of Start Codon: This interaction positions the ribosome so that the
start codon (AUG) is placed precisely in the ribosome.
 Ensures Accuracy: By aligning the ribosome correctly, it prevents translation
from starting at the wrong codon, ensuring proper protein synthesis
AUG: AUG is the start codon in both prokaryotes and eukaryotes. It signals the
ribosome where to begin translating the mRNA into a protein.
Stop codon: UAA, UGA, UAG
“universal codon” usually refers to the idea that the genetic code—the set of
rules by which codons (triplets of nucleotides) specify amino acids—is universal
across almost all living organisms. In other words, the same codon generally codes
for the same amino acid in bacteria, plants, animals, and humans.
3. RpoH mRNA – Stem I Regulation
Stem I is a secondary structure in the 5′ untranslated region (UTR) of the RpoH
mRNA.
This stem-loop occludes the ribosome binding site (RBS), including the Shine-
Dalgarno (SD) sequence and AUG start codon.
As a result, ribosomes cannot bind efficiently, leading to weak translation.
Regulation occurs when environmental or cellular signals cause stem I to unfold or
be disrupted, exposing the SD and AUG.
Once exposed, ribosomes can bind, resulting in strong translation

RpoS mRNA – Small RNA Regulation

RpoS mRNA exists in two states:
 (a) Weak translation: The SD sequence and AUG are hidden within a stem-loop,
preventing ribosome access.
(b) Strong translation: A small regulatory RNA (sRNA) binds to the mRNA and
disrupts the inhibitory structure.
This sRNA binding exposes the SD and AUG, allowing ribosomes to initiate
translation efficiently.
RpoH (σ³²) is a sigma factor in Escherichia coli that activates transcription of heat
shock genes, helping the cell respond to stress like elevated temperature.
The rpoH gene produces RpoH mRNA, which must be translated into the σ³² protein
to initiate this stress response
The regulation of RpoH mRNA occurs post-transcriptionally, mainly through RNA
secondary structures in its 5′ untranslated region (5′ UTR):
Stem I Structure
 Stem I is a hairpin loop that forms near the ribosome binding site (RBS),
including the Shine-Dalgarno (SD) sequence and start codon (AUG).
 When Stem I is folded, it blocks ribosome access, leading to weak translation.
 Under heat shock or stress conditions, cellular factors (like RNA chaperones or
temperature-induced melting) can disrupt Stem I, exposing the SD and AUG.
 This allows ribosomes to bind, resulting in strong translation of RpoH mRNA
into σ³² protein.
4. Translational Elongation requires three steps: (1) EF-Tu and GTP binds an aminoacyl
t-RNA to the ribosomal A site (this is coming of the second amino acid). (2) Peptidyl
transferase forms a peptide bond between the peptide in the P site and the newly
arrived aminoacyl t-RNA in the A site. (3) EF-G with GTP translocate the growing
peptidyl t-RNA with its mRNA codon to the P site.
side notes:
EF-Tu delivers aminoacyl-tRNAs (tRNAs charged with amino acids) to the
ribosome’s A site during protein synthesis.
Only if the codon–anticodon pairing is correct does EF-Tu hydrolyze GTP to GDP,
releasing the tRNA into the ribosome.
EF-G is a GTP-binding protein that drives translocation — the movement of the
ribosome along the mRNA by one codon after a peptide bond has formed
Mechanism:
1. After peptidyl transferase links the new amino acid, the ribosome has peptidyl-
tRNA in the A site and deacylated tRNA in the P site.
2. EF-G–GTP binds to the ribosome.
3. Hydrolysis of GTP provides the energy to shift the ribosome forward by one
codon.
4. This moves the peptidyl-tRNA into the P site and ejects the empty tRNA from
the E site.

5. Puromycin assay
Puromycin mimics tyrosyl t-RNA which only binds to A site of the ribosome
 If puromycin can bind to A site, it will form peptide bond with the amino acid in the P
site, and form puro-peptide (which we measure in the experiment)
If there is another tRNA binds in A site ribosome, then puromycin cannot bind to A
site, therefore puro-peptide cannot be formed.

Side notes:
amino acid – tRNA
fmet – tRNA fmet
met – tRNA met
met – tRNA fmet

a) when tRNA fmet which carry fmet is used with the presence of ribosome, then the
concentration of puro-peptide is increased (red graph), in the other side when
ribosome is absent, puro-peptide is relatively low. Conclusion: fmet - tRNA fmet
binds to ribosome in the P site so the puromycin can bind to A site and form puro-
peptide
b) when tRNA met which carry met is used either with the presence of ribosome or
absent of ribosome the concentration of puro-peptide is low. Conclusion: met -
tRNA met binds to ribosome in the A site so the puromycin cannot bind thus puro-
peptide cannot be formed
c) when tRNA fmet which carry met is used with the presence of ribosome, the
concentration of puro-peptide is increased, but when ribosome is absent puro-
peptide is relatively low. Conclusion: fmet - tRNA fmet binds to ribosome in the P
site so the puromycin can bind to A site and form puro-peptide
6. EF-T roles
 Panel (a): EF-Tu–GDP as Substrate
 Observation:
With EF-Ts (+EF-Ts): Rapid and significant formation of the ternary complex.
Without EF-Ts (–EF-Ts): Very little complex formation.
 Interpretation:
EF-Ts is essential when EF-Tu is in its GDP-bound form. EF-Ts acts as a guanine
nucleotide exchange factor (GEF), helping EF-Tu release GDP and bind GTP,
which is required for forming the active ternary complex with aminoacyl-tRNA
(aa-tRNA). Without EF-Ts, EF-Tu–GDP remains inactive.
Panel (b): EF-Tu–GTP as Substrate
 Observation:
With EF-Ts (+EF-Ts): Rapid and significant formation of the ternary complex.
Without EF-Ts (–EF-Ts): Very little complex formation.
 Interpretation:
EF-Ts is essential when EF-Tu is in its GDP-bound form. EF-Ts acts as a guanine
nucleotide exchange factor (GEF), helping EF-Tu release GDP and bind GTP,
which is required for forming the active ternary complex with aminoacyl-tRNA
(aa-tRNA). Without EF-Ts, EF-Tu–GDP remains inactive.
Panel (c): EF-Tu + GTP as Substrate
 Observation:
Again, both +EF-Ts and –EF-Ts conditions show similar complex formation.
 Interpretation:
EF-Tu can spontaneously bind GTP in vitro without EF-Ts assistance. Once GTP
is bound, EF-Tu becomes active and can form the ternary complex. This shows that
 EF-Tu has intrinsic ability to exchange GDP for GTP under these conditions,
though in vivo EF-Ts enhances this process.

7. eIF4A
 Function: An ATP-dependent RNA helicase.
 Role: Unwinds secondary structures in the 5′ untranslated region (UTR) of mRNA,
allowing the ribosome to scan smoothly toward the start codon.
 Complex: Part of the eIF4F complex (with eIF4E and eIF4G).
eIF4G
 Function: A large scaffold protein.
 Role: Bridges multiple components:
o Binds eIF4E (cap-binding protein) and eIF4A.
o Connects the mRNA to the 43S pre-initiation complex (small ribosomal
subunit + Met-tRNAi).
 Importance: Central organizer that recruits the ribosome to the mRNA’s 5′ cap.
eIF1
 Function: Fidelity factor for start codon recognition.
 Role: Ensures that the ribosome does not initiate at non-AUG codons.
 Mechanism: Promotes scanning until the correct AUG is found; released once the
start codon is recognized.
eIF1A
 Function: Stabilizer of the pre-initiation complex.
 Role: Works with eIF1 to promote scanning and proper positioning of Met-tRNAi in
the P site.
 Additional Role: Helps in the transition from the 43S pre-initiation complex to the
48S complex, preparing for 60S subunit joining.
8. The experiment is trying to demonstrate whether peptidyl transferase activity—the
ribosome's ability to catalyze peptide bond formation—is dependent on ribosomal
proteins, or whether it can persist even after proteins are removed.
Experimental Setup
- Ribosomes from E. coli (E70S and E50S) and Thermus aquaticus (T50S) were
treated with:
- SDS: denatures proteins
- Proteinase K (PK): digests proteins
- Phenol: removes proteins and denatures nucleic acids
- The treated ribosomes were tested for peptidyl transferase activity using a fragment
reaction that produces fMet-puromycin, detectable by autoradiography.
Key Observations
- Lane 1 (E70S untreated): Strong signal → active peptidyl transferase.
- Lane 2–4 (E. coli treated): Signal disappears after SDS + PK or phenol → activity
lost.
- Lane 5 (T50S untreated): Strong signal → active.
- Lane 6 (T50S + SDS + PK): Still shows activity → resistant to protein removal.
- Lane 7 (T50S + phenol): Still shows activity → resistant to protein removal.
 - Lane 8 (T50S + SDS + PK + phenol): Still shows activity → resistant to protein
removal.
- Lane 9 (no ribosomes): No signal → negative control.
Interpretation
- E. coli ribosomes lose activity when proteins are removed → suggests proteins are
essential or rRNA is more fragile.
- T. aquaticus ribosomes retain activity after protein removal → implies rRNA alone
can catalyze peptide bond formation, especially in thermophilic species with more
stable RNA.
- Phenol treatment abolishes activity in all cases → confirms that rRNA integrity is
crucial.
9. tmRNA stands for transfer-messenger RNA.
It is a special RNA molecule found in bacteria that has properties of both tRNA and
mRNA.
Structurally, tmRNA has:
A tRNA-like domain (TLD) that can be charged with alanine.
An mRNA-like region that encodes a short peptide tag.
Role of tmRNA in Translation
 tmRNA rescues ribosomes that get stalled on damaged or incomplete mRNAs (called
non-stop mRNAs).
 When a ribosome reaches the end of an mRNA without encountering a stop codon, it
cannot release properly.
 tmRNA enters the ribosome’s A site, acting like a tRNA:
1. Donates its alanine to the growing polypeptide.
2. Provides a short mRNA template that encodes a peptide tag.
3. This tag is added to the incomplete protein.
4. The tag directs the faulty protein for degradation by proteases.
5. Meanwhile, the ribosome is freed to engage in new rounds of translation.
Why Bacteria Need tmRNA
 Ribosome rescue: Prevents ribosomes from being trapped on defective mRNAs.
 Quality control: Marks incomplete or faulty proteins with a degradation tag so they
don’t accumulate.
 Survival advantage: Ensures efficient recycling of ribosomes and maintains protein
homeostasis, especially under stress conditions where mRNA damage is common.
10. Experiment
11. The major DNA polymerase for replication in E. coli is DNA polymerase III
holoenzyme, while the gap-filling polymerase for repair is DNA polymerase I. Pol
III drives high-speed, high-fidelity chromosome replication, whereas Pol I removes
RNA primers and fills short gaps during repair processes.
Activity of Pol III:
 High processivity: Can synthesize >50 kb of DNA per binding event.
 High speed: Extends DNA at ~1000 nucleotides per second.
 Proofreading: Possesses 3′→5′ exonuclease activity to correct misincorporated
bases, ensuring replication fidelity.
 Activity of Pol I:
 5′→3′ exonuclease activity: Removes RNA primers from Okazaki fragments
and excises damaged nucleotides.
 DNA synthesis: Fills gaps with DNA after primer removal or excision repair.
 3′→5′ exonuclease activity: Provides proofreading during gap filling.

12. Unidirectional Replication

 Meaning: DNA replication proceeds in one direction only from the origin of
replication.
 Mechanism: A single replication fork is formed at the origin, and DNA
synthesis moves away from that point in one direction.
 Example: Some plasmids and viruses replicate unidirectionally.
 Result: Only one replication fork is active, so replication is slower compared
to bidirectional replication.
Bi-directional Replication
 Meaning: DNA replication proceeds in two opposite directions from the origin
of replication.
 Mechanism: Two replication forks are formed at the origin, moving away from
each other.
 Example: E. coli and most eukaryotic chromosomes replicate bidirectionally.
 Result: Replication is faster and more efficient because both forks synthesize
DNA simultaneously, covering more of the genome in less time.
13. Okazaki fragments are short DNA pieces synthesized on the lagging strand.
The leading strand is the DNA strand synthesized continuously in the same direction as
the replication fork movement (5′→3′ direction). Only one primer is needed at the origin,
and synthesis continues smoothly.
The lagging strand is The DNA strand synthesized discontinuously, opposite to the fork
movement. Because DNA polymerases can only extend in the 5 ′→3 ′ direction, replication
occurs in short bursts (Okazaki fragments), later joined by DNA ligase.
14. Roles of SSB (Single stranded DNA binding protein):
 Binding to ssDNA: SSB binds tightly and cooperatively to single-stranded
DNA regions exposed during replication (aid for helicase to interact with
DNA strand), repair, and recombination
 Prevents secondary structures: Keeps ssDNA from forming hairpins or
other secondary structures that would block polymerase activity (inhibit the
DNA to anneal with its complement strand).
 Protection: Shields ssDNA from nucleases and chemical damage.
Roles of DnaB (Replicative helicase)
ring-shaped hexameric helicase protein in bacteria, acting as the main motor
enzyme that unwinds double-stranded DNA (dsDNA) into single strands
(ssDNA) at the replication fork, using ATP hydrolysis to power its movement along
the DNA, enabling replication to proceed
15. Experiment
16. NER-DNA repair
 Eukaryotic cells use two types of nucleotide excision repair (NER): Global Genome
NER (GG-NER), which scans the entire genome for DNA damage, and
Transcription-Coupled NER (TC-NER), which specifically targets lesions that
block transcription in actively expressed genes
- GG-NER
Scope: Operates throughout the entire genome, even in non-transcribed regions.
Damage recognition: Specialized proteins (like XPC complex in humans) detect
bulky DNA lesions such as UV-induced thymine dimers or chemical adducts.
Process:
Damage recognition by XPC and associated factors.
Recruitment of TFIIH complex, which unwinds DNA around the lesion.
Endonucleases (XPF-ERCC1 and XPG) cut out a ~24–32 nucleotide segment
containing the damage.
DNA polymerase fills the gap, and DNA ligase seals it.
- TC-NER
Scope: Focuses on actively transcribed genes.
Damage recognition: Triggered when RNA polymerase II stalls at a lesion during
transcription.
Process:
Stalled RNA polymerase signals repair machinery.
Proteins like CSA and CSB recognize the stalled complex.
Recruitment of TFIIH and other NER factors follows, similar to GG-NER.
Damaged DNA is excised, resynthesized, and ligated.
17. Panel (a)
- Chromosome: Contains the Ds element and the C gene (responsible for pigment).
- Phenotype: Solid-colored oval, indicating normal pigmentation.
- Explanation: Ds is present but inactive on its own. Without Ac, Ds cannot move.
The C gene remains functional, so pigment is produced.

Panel (b)
- Chromosome: Ds has inserted into the C gene, disrupting its function.
- Phenotype: Solid-colored oval again, but labeled Mutant C.
- Explanation: Ds insertion mutates the C gene, preventing pigment production.
However, since Ac is still absent, Ds remains in place, and the mutation is stable.

Panel (c)
- Chromosome: Both Ac and Ds are present.
- Phenotype: Speckled oval, indicating variegated pigmentation.
- Explanation: Ac provides the transposase enzyme that allows Ds to move out of the
C gene during development. This restores C gene function in some cells, leading to
patches of pigment—a classic example of somatic reversion.
18. Mechanism of bacteria transposon
Cut and paste (non-replicative)
Process:
 - The transposon is excised from its original DNA location by the enzyme
transposase.
- It is then inserted directly into a new target site.
Key Features:
- No copy of the transposon is left behind at the original site.
- The genome size remains unchanged.
- Example: Tn10 in bacteria.

Replicative transposon
Process:
- The transposon is replicated during the transposition event.
- One copy stays at the original site, while another copy is inserted into a new site.
Key Features:
- Genome size increases because the transposon is duplicated.
- This mechanism can spread transposons widely across the genome.
- Example: Tn3 family transposons.
19. Structure of an antibody
Each antibody is made of: 2 heavy chains and 2 light chains

To make one antibody, a B cell must choose:

1 type of heavy chain
1 type of light chain

So total antibody diversity =

(numbers of heavy-chain combinations) × (number of light-chain combinations)

- Heavy chain combination (1 heavy chain is made up of V-D-J)

Heavy chains are made by combining:

 48 V (variable) segments
 23 D (diversity) segments
 6 J (joining) segments

Heavy chain combinations = 48×23×6 = 6624

- Light chain combination (1 light chain is made up of either lambda or kappa)

two types of light chain:

 λ (lambda): 170 options

 κ (kappa): 205 options

light chain combination = 170 + 275 = 375

 Each antibody uses 1 heavy chain combination and 1 light chain combination

Total antibody diversity = 6624 x 375 = 2,484,000

20. (i) c
(ii) d
(iii) f
(iv) e
(v) g
When DNA is damaged—especially by UV light—a common lesion forms:
Cyclobutane pyrimidine dimer
(e.g., thymine–thymine dimer)
Normal DNA polymerases stall when they encounter this, causing replication to
stop.
But polymerase η (Pol η) can:
 insert the correct bases (A–A) across a thymine dimer,
 continue replication even though the DNA is damaged.
This prevents the replication fork from collapsing

21. Trigger factor

- Trigger factor is a ribosome-associated chaperone in bacteria.
- It binds near the ribosomal exit tunnel on the large ribosomal subunit.
- Its role is to catch the nascent polypeptide as it emerges and shield hydrophobic
regions from water, preventing premature folding or aggregation.
- It forms a hydrophobic cradle or basket that temporarily holds the growing
polypeptide chain.
22. True
- Wobble base pairing refers to the flexible pairing between the third base of the
mRNA codon and the first base of the tRNA anticodon.
- This flexibility allows one tRNA to recognize multiple codons, contributing to the
degeneracy of the genetic code.
- It typically involves non-standard base pairs, such as:
- G–U
- Inosine (I) pairing with U, C, or A

Kodon mRNA : AUG

Antikodon tRNA: UAC
23. EF-Tu
24. D
25. False
Prokaryotic mRNAs generally have a shorter lifetime — often just a few minutes.
- This rapid turnover allows bacteria to quickly adjust gene expression in response to
environmental changes.
- However, because mRNA is short-lived, translational control is limited.

Eukaryotic mRNAs tend to be more stable, lasting from hours to days.

- This longer lifespan allows more opportunities for regulation at the translational
level, such as:
- mRNA localization
- microRNA-mediated repression
- ribosome pausing or selective translation