Biochem2Sem5

1. Describe Meselson and Stahl experiment in detail. (10

Marks)

The Meselson and Stahl experiment (1958) is a classic

experiment that demonstrated the semi-conservative nature of
DNA replication. This experiment provided direct evidence for
how DNA replicates, showing that each daughter molecule
contains one parental strand and one newly synthesized strand.

1. Objective: To understand the mechanism of DNA replication

and determine which model (conservative, semi-conservative, or
dispersive) describes it accurately.

2. Conservative Model Hypothesis: In this model, the parental

DNA remains intact, and an entirely new molecule is
synthesized.

3. Semi-Conservative Model Hypothesis: The parental DNA

strands separate, each serving as a template for a new
complementary strand, resulting in daughter DNA with one old
and one new strand.

4. Dispersive Model Hypothesis: DNA replication involves

fragmentation, with each fragment of the parent molecule
replicated separately, creating hybrid molecules with mixed new
and old DNA.

5. Choice of Organism: E. coli was selected for this experiment

due to its simple DNA structure and ease of handling.

6. Isotope Labeling: Meselson and Stahl used nitrogen isotopes,

N-15 (heavy nitrogen) and N-14 (light nitrogen), to label DNA.
Initially, E. coli was grown in N-15 to label its DNA.

7. Transfer to N-14 Medium: After labeling DNA with N-15, the

bacteria were transferred to a medium containing N-14. This
shift allowed tracking of the "old" and "new" DNA strands
during replication.

8. Density Gradient Centrifugation: The DNA samples were

subjected to density gradient centrifugation in cesium chloride
(CsCl) solution, separating DNA by density.

9. Round 1 Observation: After one round of replication in N-14,

a single intermediate-density band appeared, indicating that each
DNA molecule contained one old (N-15) and one new (N-14)
strand.

10. Round 2 Observation: After the second round of replication,

two distinct bands were observed: one intermediate-density band
(hybrid) and one lighter band, confirming semi-conservative
replication.
 11. Conclusion: The experiment supported the semi-
conservative model of DNA replication, where each new DNA
molecule has one parent strand and one new strand.

12. Exclusion of Conservative Model: The appearance of

intermediate-density DNA in the first generation ruled out the
conservative model, which would have shown separate bands
for heavy and light DNA.

13. Exclusion of Dispersive Model: The presence of distinct

bands in the second generation ruled out the dispersive model,
which would have resulted in a single, gradually lighter band
over generations.

14. Significance: This experiment provided conclusive evidence

for semi-conservative replication, a fundamental concept in
molecular biology that explains genetic inheritance.

15. Biological Implication: The semi-conservative replication

model ensures stability of genetic information across
generations, reducing errors and maintaining the integrity of
genetic material.

1.(a) Write a note on initiation of DNA replication in

prokaryotes. (5 Marks)
1. Definition: DNA replication initiation is the beginning phase
of DNA synthesis, starting at specific sites called origins of
replication.

2. Origin of Replication (OriC): In E. coli, replication begins at a

unique sequence called OriC, containing about 245 base pairs
with specific DNA sequences.

3. DnaA Protein Binding: Initiation begins with DnaA proteins

binding to the DnaA-boxes within the OriC, unwinding the
DNA at the origin.

4. Helicase Loading: DnaC protein loads the DnaB helicase onto

the DNA, allowing the DNA double helix to be unwound.

5. Formation of Replication Bubble: Helicase unwinding

generates a replication bubble at the OriC, creating single-
stranded regions for replication.

6. Primase Action: Primase enzyme synthesizes short RNA

primers on the single-stranded DNA, providing a starting point
for DNA polymerase.

7. DNA Polymerase III: DNA Polymerase III binds to the RNA

primer and initiates DNA synthesis in the 5' to 3' direction.
 8. Bidirectional Replication: From the origin, two replication
forks move in opposite directions, enabling simultaneous DNA
replication on both strands.

9. Regulation: Replication initiation is tightly regulated to occur

once per cell cycle, ensuring only one complete genome copy
per cell division.

10. Importance: Accurate initiation is essential for faithful DNA

replication, maintaining genomic stability in rapidly dividing
prokaryotic cells.

1.(b) Describe rolling circle model of DNA Replication. (5

Marks)

1. Definition: The rolling circle model is a mechanism of DNA

replication commonly used by plasmids, certain viruses, and
bacteriophages with circular DNA.
2. Initiation: A single-strand break occurs on one of the circular
DNA strands, providing a free 3’-OH end for elongation.

3. Polymerization: DNA polymerase begins synthesis at the 3’-

OH end, using the unbroken strand as a template, displacing the
5' end as replication proceeds.
 4. Formation of Single-Stranded Tail: The displaced single
strand forms a tail as replication progresses around the circular
DNA template.

5. Continuous Synthesis: The leading strand is synthesized

continuously as DNA polymerase follows the circle, moving in a
loop-like fashion.

6. Displaced Strand Replication: The displaced single strand can

undergo complementary synthesis, either forming a circular
structure or multiple tandem copies.

7. Repetition: The cycle can continue, producing multiple copies

of the circular DNA rapidly, advantageous for viral genomes
that replicate in host cells.

8. Applications: Commonly observed in bacteriophage lambda

and plasmid replication, where rapid DNA amplification is
beneficial.

9. Advantages: This replication mode allows for quick DNA

replication, supporting rapid viral proliferation within host cells.

10. Significance: The rolling circle model exemplifies an

efficient replication strategy for small circular genomes,
highlighting DNA’s adaptability across various organisms.
1. Give a detailed account of the semi-conservative model of
replication with experimental evidence. (10 Marks)

The semi-conservative model of DNA replication was first

proposed by Watson and Crick and later confirmed by the
Meselson-Stahl experiment in 1958. This model describes how
each strand of the DNA double helix serves as a template for a
new complementary strand during replication.

1. Definition of Semi-Conservative Replication:

Semi-conservative replication involves each parental DNA
strand acting as a template to synthesize a new complementary
strand.

2. Proposal by Watson and Crick:

Watson and Crick suggested the semi-conservative model after
discovering DNA's double-helix structure, hypothesizing that
one strand from each parent molecule would be preserved in
each daughter molecule.

3. Mechanism of Replication:
During replication, the DNA double helix unwinds, and each
parental strand pairs with new complementary nucleotides,
resulting in two DNA molecules with one old and one new
strand.
 4. Supporting Experiment - Meselson and Stahl:
Meselson and Stahl conducted their experiment using E. coli to
demonstrate that DNA replication is semi-conservative.

5. Experimental Design:
E. coli was grown in a medium with N-15, then switched to a
medium with N-14 to distinguish "old" and "new" DNA strands
by density.

6. Density Gradient Centrifugation:

Meselson and Stahl used density gradient centrifugation in
cesium chloride (CsCl) to separate DNA based on density
differences.

7. Round 1 Results:
After the first round of replication, they observed an
intermediate-density band, showing that each DNA molecule
contained one heavy and one light strand.

8. Round 2 Results:
In the second generation, two bands appeared: one intermediate
(hybrid) and one light, confirming the semi-conservative nature
of replication.

9. Exclusion of Conservative Model:

The intermediate band after one round ruled out the conservative
model, which would have shown separate bands for old and new
DNA.
 10. Exclusion of Dispersive Model:
The two bands after the second round ruled out the dispersive
model, which would have shown a single, gradually lighter
band.

11. Biological Significance:

Semi-conservative replication ensures that genetic information is
accurately passed from one cell generation to the next, reducing
mutation rates.

12. Genome Stability:

By preserving one original strand, semi-conservative replication
helps maintain DNA integrity, supporting cellular stability.

13. Enzyme Involvement:

Enzymes like DNA helicase, primase, and DNA polymerase are
involved, ensuring a smooth replication process.

14. Evidence in Other Organisms:

The semi-conservative model applies to both prokaryotic and
eukaryotic cells, showing the universality of DNA replication.

15. Legacy of the Experiment:

The Meselson-Stahl experiment is considered one of the most
elegant experiments in biology, proving a central concept of
genetics and molecular biology.
 1.(a) Theta (θ) Model of Replication. (5 Marks)

The theta (θ) model of replication is a type of bidirectional DNA

replication observed primarily in circular DNA, such as bacterial
genomes.

1. Definition: The θ model of replication occurs in circular DNA

molecules, forming a structure resembling the Greek letter theta
(θ) as replication progresses.

2. Origin of Replication (OriC): Replication begins at a specific

origin in the circular DNA molecule, where the double helix
opens up.

3. Bidirectional Replication: Two replication forks move in

opposite directions from the origin, allowing simultaneous
synthesis on both sides.

4. Formation of Theta Structure: As replication progresses, the

circular DNA forms an intermediate theta-like structure, hence
the name θ model.

5. Leading and Lagging Strands: Each fork has a leading strand,

synthesized continuously, and a lagging strand, synthesized
discontinuously in Okazaki fragments.
 6. Single Replicon: Since prokaryotic genomes typically have a
single origin, the entire circular genome forms one replicon (unit
of replication).

7. Completion of Replication: When the two forks meet on the

opposite side of the circle, replication concludes, resulting in
two circular DNA molecules.

8. Applications: Commonly seen in bacterial chromosomes and

some plasmids, the θ model is efficient for replicating small,
circular genomes.

9. Speed and Efficiency: The bidirectional mechanism allows

rapid replication, a necessity for fast-growing bacterial cells.

10. Significance: The θ model ensures the accuracy and speed of

replication, critical for prokaryotic cells with high division rates.

1.(b) Discuss DNA replication initiation in E. coli. (5 Marks)

The initiation of DNA replication in E. coli involves the origin

of replication, OriC, and several proteins that facilitate the
unwinding and preparation of DNA for synthesis.

1. OriC - Origin of Replication: DNA replication starts at OriC,

a specific 245 base-pair sequence on the E. coli chromosome.
 2. DnaA Protein Binding: DnaA proteins bind to DnaA-boxes
within OriC, causing DNA unwinding at the origin.

3. DNA Unwinding: The binding of DnaA creates an open

complex, where the DNA double helix begins to unwind,
forming a small replication bubble.

4. Loading of Helicase (DnaB): DnaC loads DnaB helicase onto

the DNA, further unwinding the helix to extend the replication
bubble.

5. Role of Primase: Primase (DnaG) synthesizes a short RNA

primer on the single-stranded DNA, which DNA polymerase
will later use as a starting point.

6. Loading of DNA Polymerase III: DNA Polymerase III, the

main enzyme for synthesis, binds to the primer and initiates
elongation in the 5’ to 3’ direction.

7. Bi-directional Replication Forks: The initiation process

establishes two replication forks that move in opposite
directions, allowing for simultaneous replication.

8. Tight Regulation: Initiation is highly regulated, ensuring that

replication only occurs once per cell cycle to prevent over-
replication.
 9. Role of DNA Gyrase: DNA gyrase relieves supercoiling
tension that forms ahead of the replication forks, maintaining
DNA stability during replication.

10. Significance in Cell Cycle: Proper initiation ensures accurate

duplication of the E. coli genome, essential for cell division and
population growth.

1. (a) Discuss the termination process of DNA replication. (5

Marks)

The termination of DNA replication involves the completion and

disassembly of the replication machinery, ensuring the
replicated chromosomes are properly segregated.

1. Definition: Termination of replication is the process that

concludes DNA synthesis, ensuring both daughter DNA
molecules are fully replicated.

2. Termination Sites (Ter Sites): In E. coli, specific termination

sequences called Ter sites are positioned opposite the origin of
replication on the circular chromosome.

3. Tus Proteins: The Ter sites bind Tus proteins, which halt the
progress of replication forks, effectively stopping DNA
polymerase activity.
4. Meeting of Replication Forks: The two replication forks meet
at the Ter sites, where Tus-Ter complexes prevent further
elongation.

5. Decatenation by Topoisomerase IV: The replicated circular

chromosomes are interlinked; topoisomerase IV introduces
temporary breaks to separate the linked DNA circles.

6. Resolution of Catenanes: The decatenation process ensures

each daughter cell receives a complete and unlinked
chromosome.

7. Disassembly of Replication Machinery: The replication

proteins detach, and any remaining Okazaki fragments on the
lagging strand are joined by DNA ligase.

8. Repair of DNA Ends: Exonuclease and repair enzymes check

and repair any mismatches or gaps to ensure integrity.

9. Separation of Chromosomes: The two chromosomes are

guided to opposite poles of the cell, preparing the cell for
division.

10. Importance of Termination: Accurate termination is essential

to prevent re-replication or incomplete replication, which could
lead to genomic instability.
1. Give a detailed account of elongation and termination of
DNA replication in E. coli. (10 Marks)

In E. coli, elongation and termination are crucial stages in DNA

replication, where the replication forks progress along the DNA
and synthesis is completed at specific termination sites.

1. Elongation Process: Elongation occurs after initiation,

involving continuous addition of nucleotides to synthesize new
DNA strands.

2. DNA Polymerase III: The primary enzyme for elongation,

DNA polymerase III, synthesizes new DNA in the 5’ to 3’
direction, attaching nucleotides complementary to the template
strand.

3. Leading and Lagging Strands: The leading strand is

synthesized continuously, while the lagging strand is
synthesized discontinuously in short Okazaki fragments.

4. Okazaki Fragments: On the lagging strand, DNA polymerase

III creates Okazaki fragments, which are later joined by DNA
ligase.

5. RNA Primers: RNA primers are synthesized by primase to

initiate Okazaki fragment formation on the lagging strand.
 6. Proofreading by DNA Polymerase III: DNA polymerase III
has proofreading activity, ensuring high fidelity by correcting
misincorporated nucleotides during elongation.

7. DNA Polymerase I and Primer Removal: DNA polymerase I

replaces the RNA primers with DNA nucleotides, filling gaps on
the lagging strand.

8. DNA Ligase: DNA ligase seals the nicks between Okazaki

fragments, creating a continuous strand on the lagging side.

9. Termination Sites (Ter Sites): Termination occurs when the

two replication forks meet at designated termination sites (Ter
sites) on the opposite side of the circular chromosome.

10. Role of Tus Proteins: Tus proteins bind to Ter sites and
prevent replication forks from progressing, ensuring replication
halts at the correct location.

11. Resolution of Catenanes: As the circular chromosomes are

interlinked (catenated) after replication, topoisomerase IV
introduces breaks to resolve these links, separating the daughter
chromosomes.

12. Disassembly of Replication Machinery: DNA polymerases

and other proteins detach from the DNA after completing
synthesis, ending replication.
 13. Final DNA Repair: Any remaining gaps or mismatches are
repaired to ensure the integrity of the new DNA.

14. Segregation: The two fully replicated and separated

chromosomes are then positioned to opposite poles of the cell,
ready for cell division.

15. Significance: Accurate elongation and termination ensure the

complete and error-free duplication of the genome, crucial for
maintaining genetic stability in bacterial populations.

1.(a) Write a note on the "Origin of Replication". (5 Marks)

The origin of replication, often referred to as "OriC" in E. coli,

is the specific sequence where DNA replication begins,
initiating the complex process of DNA duplication.

1. Definition: The origin of replication is a specific DNA

sequence where replication initiates, marking the start of DNA
synthesis.

2. OriC in E. coli: In E. coli, the origin is called OriC, a 245

base-pair segment that contains specific DNA sequences
recognized by initiation proteins.
 3. DnaA Binding: The DnaA protein binds to DnaA-boxes
within OriC, causing localized DNA unwinding to create an
open complex.

4. AT-Rich Region: OriC contains an AT-rich region, which is

easier to unwind due to the lower stability of A-T base pairs
compared to G-C pairs.

5. Formation of Replication Bubble: Unwinding at OriC creates

a replication bubble, with single-stranded regions that serve as
templates for new strand synthesis.

6. DnaB Helicase Loading: DnaC protein assists in loading

DnaB helicase onto the open DNA, which further unwinds the
helix, expanding the replication bubble.

7. Role of Primase: Primase synthesizes RNA primers on the

exposed single-stranded DNA at OriC, providing starting points
for DNA polymerase.

8. Bidirectional Forks: From the origin, two replication forks

move in opposite directions, allowing simultaneous replication
on both sides of the circular DNA.

9. Regulation: OriC activity is tightly regulated to prevent re-

initiation before cell division, ensuring only one complete
replication per cell cycle.
 10. Importance: The origin of replication is essential for the
controlled start of DNA synthesis, crucial for genomic stability
and proper cell division in prokaryotes.

1. Write notes on: (a) Okazaki Fragments. (5 Marks)

Okazaki fragments are short DNA segments synthesized on the

lagging strand during DNA replication, playing a critical role in
ensuring the complete replication of both DNA strands.

1. Definition: Okazaki fragments are short DNA segments

produced on the lagging strand during DNA replication.

2. Discontinuous Synthesis: On the lagging strand, synthesis is

discontinuous because DNA polymerase can only add
nucleotides in the 5’ to 3’ direction.

3. Length of Fragments: In prokaryotes, Okazaki fragments are

typically 1,000 to 2,000 nucleotides long, while in eukaryotes,
they are shorter, about 100 to 200 nucleotides.

4. Role of Primase: Primase synthesizes a short RNA primer for

each Okazaki fragment, providing a 3’-OH end for DNA
polymerase to initiate synthesis.
 5. DNA Polymerase III: DNA polymerase III extends each
Okazaki fragment by adding nucleotides to the primer,
synthesizing DNA in the 5’ to 3’ direction.

6. Replacement of RNA Primers: DNA polymerase I replaces

RNA primers with DNA nucleotides, leaving gaps between
Okazaki fragments.

7. DNA Ligase Activity: DNA ligase seals the nicks between

Okazaki fragments, joining them into a continuous DNA strand
on the lagging side.

8. High Fidelity: The combined actions of polymerase

proofreading and ligase ensure that the lagging strand is
synthesized accurately and without gaps.

9. Significance: Okazaki fragments enable the complete and

accurate synthesis of the lagging strand, essential for genome
stability.

10. Named After: They were discovered by Reiji and Tsuneko

Okazaki in the 1960s, who provided insights into the mechanism
of lagging strand synthesis.

2.Write notes on:

 (a) MMR (Mismatch Repair) (5 Marks)

Mismatch repair (MMR) is a DNA repair mechanism that

corrects errors introduced during DNA replication, specifically
mismatches that escape proofreading by DNA polymerase.

1. Definition: Mismatch repair (MMR) is a post-replication

repair mechanism that corrects base mismatches in newly
synthesized DNA strands.

2. Detection of Mismatches: MMR detects incorrect base

pairings (such as A-G or T-C mismatches) that occur due to
replication errors.

3. Identification of New Strand: The newly synthesized strand is

identified by MMR machinery, often through unmethylated
adenines in GATC sequences in prokaryotes.

4. Key Proteins (Mut Proteins): In E. coli, proteins MutS, MutL,

and MutH recognize mismatches, excise the error, and fill the
gap.
5. Role of MutS: MutS binds to the mismatch site, initiating the
repair process.

6. MutL and MutH Action: MutL stabilizes the complex, while

MutH makes a cut in the new DNA strand to remove the
mismatch.

7. Exonuclease Action: Exonucleases degrade the DNA around

the mismatch, creating a single-stranded gap.

8. DNA Polymerase and Ligase: DNA polymerase resynthesizes

the correct sequence, and DNA ligase seals the nick.

9. Biological Significance: MMR significantly reduces mutation

rates, maintaining genetic stability.

10. MMR Deficiency: Defects in MMR are linked to hereditary

nonpolyposis colorectal cancer (HNPCC), highlighting its
importance in preventing mutations.

(b) NER (Nucleotide Excision Repair) (5 Marks)

 Nucleotide excision repair (NER) is a versatile DNA repair
mechanism that removes bulky lesions, such as thymine dimers
and other UV-induced damage, from DNA.

1. Definition: NER repairs bulky DNA lesions by excising

damaged nucleotides and filling the gap with new DNA.

2. Types of Lesions: NER corrects damage like UV-induced

thymine dimers and other distortions in the DNA helix.

3. Two Pathways in NER: There are two types of NER: global

genomic NER (repairs throughout the genome) and
transcription-coupled NER (repairs actively transcribed genes).

4. Damage Recognition: Damage is recognized by specific

proteins, such as UvrA and UvrB in E. coli.

5. Formation of Pre-Excision Complex: UvrA and UvrB form a

complex to detect and verify the lesion site.

6. UvrC Endonuclease: UvrC cuts the DNA strand on either side

of the lesion, excising the damaged section.
7. Removal of Damaged Segment: UvrD helicase helps remove
the excised segment, leaving a gap.

8. DNA Polymerase and Ligase: DNA polymerase fills in the

gap with the correct nucleotides, and DNA ligase seals the nick.

9. Biological Significance: NER is crucial for correcting UV-

induced damage and maintaining genome stability.

10. NER Defects: Mutations in NER genes lead to conditions

like xeroderma pigmentosum, which increases UV sensitivity
and skin cancer risk.

(c) Ames Test (5 Marks)

The Ames test is a biological assay used to assess the mutagenic

potential of chemical compounds by observing their effect on
bacterial DNA.

1. Purpose: The Ames test evaluates whether a chemical can

induce mutations in DNA, indicating potential carcinogenicity.
 2. Test Organism: It typically uses strains of Salmonella
typhimurium that have mutations preventing histidine synthesis.

3. Mutation Reversion Principle: Mutagens induce mutations

that revert the bacterial strain to histidine synthesis, allowing it
to grow on histidine-free media.

4. Addition of Rat Liver Extract: To simulate mammalian

metabolism, a rat liver enzyme mix (S9 fraction) is added,
activating some compounds.

5. Positive Control: Known mutagens are used as controls to

ensure the sensitivity of the test.

6. Quantitative and Qualitative Results: The number of revertant

colonies indicates the mutagenic strength of the test compound.

7. Simple and Cost-Effective: The Ames test is widely used in

initial screening for mutagenic chemicals due to its simplicity.
 8. Predictive Value for Carcinogenicity: Many mutagens
identified by the Ames test are also carcinogenic in animals.

9. Limitations: While useful, the test cannot fully replicate

complex mammalian metabolism, limiting its predictive
accuracy.

10. Significance: The Ames test provides an efficient way to

identify potential carcinogens and ensure chemical safety in
research and industry.

(d) BER (Base Excision Repair) (5 Marks)

Base excision repair (BER) is a DNA repair pathway that

corrects small, non-distorting lesions in DNA, often caused by
oxidative damage, deamination, or alkylation.

1. Definition: BER repairs single-base lesions that do not distort

the DNA helix, such as uracil or oxidized bases.

2. Types of Damage Repaired: BER corrects oxidative damage,

deamination, alkylation, and other small modifications to bases.
 3. Glycosylase Enzymes: DNA glycosylases recognize and
remove the damaged base, creating an abasic (AP) site.

4. AP Endonuclease: The AP site is recognized by AP

endonuclease, which makes a cut at the site, creating a single-
strand break.

5. DNA Polymerase Action: DNA polymerase fills the gap by

adding the correct base opposite the AP site.

6. DNA Ligase: DNA ligase seals the nick, restoring the DNA
strand’s integrity.

7. Short Patch and Long Patch Repair: BER can proceed via
short-patch (replacing one nucleotide) or long-patch (replacing
2-10 nucleotides) repair pathways.

8. Significance in Cellular Defense: BER plays a critical role in

defending cells against small-scale DNA damage, reducing
mutation rates.
9. Mutations in BER Pathway: Deficiencies in BER enzymes are
linked to cancer and other diseases due to accumulation of DNA
lesions.

10. Conservation Across Species: BER is conserved across

prokaryotes and eukaryotes, emphasizing its essential role in
genomic maintenance.

2. Types of DNA Damage (10 Marks)

DNA damage can result from both endogenous and exogenous

factors, leading to various alterations that affect genome stability
and cellular function.

1. Single-Strand Breaks (SSBs): Breaks in one strand of the

DNA helix, often caused by reactive oxygen species or
replication errors.

2. Double-Strand Breaks (DSBs): Breaks in both strands, highly

damaging, resulting from ionizing radiation or severe oxidative
stress.
 3. Base Modifications: Changes in bases, such as oxidation (8-
oxoG), alkylation, and deamination, can alter base-pairing
properties, leading to mutations.

4. Thymine Dimers: UV radiation can induce the formation of

covalent bonds between adjacent thymine bases, distorting the
DNA helix.

5. Pyrimidine Dimers: Similar to thymine dimers, other

pyrimidine bases like cytosine can also form dimers, disrupting
DNA structure.

6. Interstrand Crosslinks: Covalent links between two strands

inhibit separation during replication, highly mutagenic and
difficult to repair.

7. Intrastrand Crosslinks: Bonds between bases on the same

strand, often caused by agents like cisplatin, leading to structural
distortions.

8. Mismatch Errors: Incorrect base pairing (e.g., A-G or T-C)

during replication, which can escape proofreading.
 9. Chemical Adducts: Attachment of large molecules to DNA
bases (e.g., aflatoxin binding), causing bulkiness and replication
issues.

10. Depurination: Loss of a purine base (adenine or guanine)

due to spontaneous hydrolysis, creating an abasic site.

11. Depyrimidination: Loss of a pyrimidine base (cytosine or

thymine), also resulting in an abasic site.

12. Bulky Lesions: Large structural alterations caused by

environmental carcinogens, like polycyclic aromatic
hydrocarbons.

13. Oxidative Damage: Reactive oxygen species can modify

bases, sugar-phosphate backbone, or cause strand breaks.

14. Alkylation Damage: Addition of alkyl groups to DNA bases,

commonly from environmental mutagens, leading to
mismatches.
 15. Significance: Understanding DNA damage types is crucial
for studying mutation sources, cancer risk, and developing repair
therapies.

2. Briefly discuss SOS repair. (10 Marks)

The SOS repair is an emergency DNA repair mechanism in

bacteria, specifically induced in response to extensive DNA
damage. It allows bacteria to survive severe DNA damage but
may lead to increased mutation rates due to its error-prone
nature.

1. Definition of SOS Repair: The SOS response is a cellular

mechanism activated in bacteria, like E. coli, under extensive
DNA damage, initiating DNA repair processes.

2. Triggering of SOS Response: The response is triggered by the

accumulation of single-stranded DNA (ssDNA) resulting from
DNA damage or stalled replication forks.

3. Role of RecA Protein: RecA binds to ssDNA, forming a

nucleoprotein filament that activates the autocleavage of LexA
repressor, de-repressing SOS genes.
 4. LexA Repressor: Under normal conditions, LexA represses
SOS genes. Damage-induced cleavage of LexA allows SOS
gene expression.

5. SOS Genes: Over 40 genes are activated, including DNA

repair enzymes, translesion polymerases (Pol IV and Pol V), and
other repair proteins.

6. Translesion Synthesis (TLS): Specialized polymerases like

Pol IV and Pol V synthesize DNA across damaged regions,
allowing replication despite lesions.

7. Error-Prone Mechanism: Translesion polymerases lack

proofreading, leading to an error-prone repair that introduces
mutations.

8. Advantages of SOS Repair: Enables cell survival in the

presence of lethal DNA damage by allowing DNA synthesis to
continue.
9. Disadvantages of SOS Repair: Increased mutation rate due to
the lack of accuracy in translesion synthesis, which can lead to
mutagenesis.

10. Regulation of the SOS Response: Once DNA damage is

repaired, RecA activity decreases, allowing LexA levels to rise
and repress SOS genes.

11. Roles of umuC and umuD Genes: These genes encode

components of Pol V, essential for translesion synthesis across
damaged DNA.

12. RecBCD Complex: This complex is involved in repairing

double-strand breaks and enhancing homologous recombination,
another component of the SOS response.

13. Applications in Research: SOS repair is used to study

mutagenesis, antibiotic resistance, and DNA repair processes in
bacteria.

14. Evolutionary Perspective: The SOS response contributes to

genetic diversity in bacterial populations, impacting adaptability
and evolution.
 15. Biological Significance: SOS repair is crucial for bacterial
survival in adverse conditions but poses a trade-off between
survival and increased mutation rates.

2. Discuss Mismatch and Base Excision Repair of DNA. (10

Marks)

Mismatch repair (MMR) and base excision repair (BER) are

DNA repair mechanisms that correct distinct types of DNA
damage, maintaining genome integrity.

1. Mismatch Repair (MMR) Overview: MMR corrects errors

introduced during DNA replication, specifically base
mismatches and small insertion/deletion loops.

2. MMR Recognition Proteins: In bacteria, MutS recognizes

mismatches, MutL stabilizes the complex, and MutH makes cuts
on the new strand.
 3. Strand Discrimination in MMR: In E. coli, the new strand is
identified by its lack of methylation at GATC sequences,
allowing repair machinery to target it.

4. Exonuclease Activity in MMR: Exonucleases remove the

mismatched segment, followed by DNA polymerase and ligase
action to fill and seal the gap.

5. Biological Significance of MMR: MMR reduces mutation

rates by correcting replication errors, playing a crucial role in
maintaining genetic stability.

6. Diseases Associated with MMR Defects: Defective MMR is

linked to cancers like Lynch syndrome (HNPCC) due to
increased mutation rates.

7. Base Excision Repair (BER) Overview: BER targets small,

non-distorting lesions such as oxidized, alkylated, or deaminated
bases, often due to cellular metabolism.

8. Glycosylase Action in BER: DNA glycosylase identifies and

removes the damaged base, creating an apurinic/apyrimidinic
(AP) site.
9. AP Endonuclease: AP endonuclease cuts the DNA backbone
at the AP site, leaving a single-strand break.

10. Filling the Gap in BER: DNA polymerase adds the correct
nucleotide(s) opposite the lesion, followed by ligase action to
seal the nick.

11. Short-Patch vs. Long-Patch BER: Short-patch repairs a

single nucleotide, while long-patch replaces 2-10 nucleotides
around the lesion.

12. Significance of BER in Cells: BER protects against

oxidative and spontaneous base modifications, crucial for
genome stability.

13. Mutations in BER Pathway: Deficiencies in BER enzymes

like glycosylase or polymerase are linked to cancer and age-
related diseases.

14. Conservation Across Species: Both MMR and BER are

highly conserved in prokaryotes and eukaryotes, highlighting
their evolutionary importance.
15. Comparative Roles: While MMR targets replication errors,
BER focuses on spontaneous base damage, both essential for
genome fidelity.

2. Explain the concept and regulation of E. coli replication.

(10 Marks)

Replication in E. coli is a well-regulated process that ensures

accurate and complete duplication of the bacterial genome
before cell division.

1. Concept of Replication in E. coli: E. coli uses a bidirectional

replication process, starting from a single origin of replication
(OriC) on its circular chromosome.

2. Initiation at OriC: Replication initiates at the OriC site, which

is rich in AT sequences, making it easier to unwind.

3. Role of DnaA Protein: DnaA binds to OriC, causing DNA

unwinding and forming an open complex for further protein
binding.
4. Loading of DnaB Helicase: DnaC helps load DnaB helicase
onto the single-stranded DNA, unwinding the double helix for
replication.

5. Primase Action: Primase synthesizes RNA primers, which

provide starting points for DNA polymerase III to begin
replication.

6. Bidirectional Forks: Two replication forks move in opposite

directions from OriC, replicating both strands of the circular
chromosome simultaneously.

7. Leading and Lagging Strands: DNA synthesis is continuous

on the leading strand and discontinuous on the lagging strand,
forming Okazaki fragments.

8. Regulation by SeqA: SeqA binds to newly replicated,

hemimethylated DNA, delaying reinitiation by blocking DnaA
access to OriC.

9. Role of Dam Methylase: Dam methylase methylates the new

strand, signaling completion of replication and allowing
potential reinitiation in future cycles.
10. Termination at Ter Sites: Replication terminates at Ter sites
on the opposite side of the chromosome from OriC, where the
forks meet.

11. Role of Tus Protein: Tus binds to Ter sites, preventing the
progression of replication forks beyond termination points.

12. Topoisomerase IV: This enzyme separates interlinked

(catenated) daughter chromosomes, allowing for chromosome
segregation.

13. Cell Cycle Coordination: Replication is tightly linked to cell

growth and division, with regulatory mechanisms ensuring
timely initiation and completion.

14. Importance of Regulation: Proper regulation prevents

incomplete replication and DNA damage, crucial for bacterial
viability.
 15. Significance in Microbial Genetics: Understanding E. coli
replication and regulation is fundamental in studying bacterial
genetics and antibiotic targeting.

2. Describe in detail DNA polymerases. (10 Marks)

DNA polymerases are enzymes responsible for synthesizing new

DNA strands, each with distinct functions in replication and
repair.

1. Definition: DNA polymerases are enzymes that catalyze the

addition of nucleotides to a growing DNA strand, synthesizing
new DNA during replication.

2. DNA Polymerase I (Pol I): In prokaryotes, Pol I is involved in

Okazaki fragment processing and repair by removing RNA
primers and filling gaps with DNA.

3. DNA Polymerase III (Pol III): The main replicative enzyme in

E. coli, Pol III synthesizes DNA continuously on the leading
strand and in fragments on the lagging strand.
4. High Processivity of Pol III: Pol III has high processivity due
to the sliding clamp, allowing it to synthesize long stretches of
DNA efficiently.

5. Proofreading Ability: Many DNA polymerases have 3’ to 5’

exonuclease activity, enabling them to correct errors during
replication.

6. Eukaryotic Polymerases: Eukaryotes have multiple

polymerases (e.g., Pol α, δ, and ε), each specialized for different
replication stages and repair functions.

7. Pol α and Primase: Pol α, along with primase, synthesizes the

RNA-DNA primer required for initiation on both strands.

8. Pol δ and Pol ε: Pol δ synthesizes the lagging strand, while

Pol ε synthesizes the leading strand, both with high fidelity and
proofreading.

9. Translesion Synthesis Polymerases: Specialized polymerases

(e.g., Pol IV and V in E. coli) bypass lesions but lack
proofreading, often
10. Role of Pol IV and Pol V in SOS Response: In E. coli, Pol
IV and Pol V are part of the SOS repair system, allowing DNA
synthesis across damaged regions but with increased mutation
rates due to their lack of proofreading.

11. Specialized Functions: Some DNA polymerases, like Pol β

in eukaryotes, are involved in base excision repair, a pathway
that corrects small, non-distorting lesions.

12. Processivity Factor: The sliding clamp (β-clamp in E. coli,

PCNA in eukaryotes) enhances the processivity of polymerases,
allowing them to synthesize DNA over long distances without
dissociating.

13. Structural Variation: DNA polymerases have structurally

diverse domains, including palm, fingers, and thumb regions,
which interact with DNA and dNTPs for accurate synthesis.

14. Fidelity of DNA Polymerases: High-fidelity polymerases

ensure low error rates in DNA replication, critical for genetic
stability. Low-fidelity polymerases, like translesion
polymerases, are more error-prone but essential for bypassing
DNA lesions.
15. Significance in Biotechnology: DNA polymerases are widely
used in PCR and DNA sequencing technologies, as well as in
molecular cloning and research applications where DNA
synthesis is essential.

3. Describe prokaryotic transcription initiation in detail. (10

Marks)

Prokaryotic transcription initiation is a key phase in gene

expression, where RNA synthesis is initiated at a specific region
of DNA. The process includes binding of RNA polymerase to
the promoter, unwinding of DNA, and formation of the first
phosphodiester bond.

1. Definition of Transcription Initiation: Transcription initiation

in prokaryotes is the process where RNA polymerase binds to
the promoter region on DNA and starts synthesizing RNA.

2. RNA Polymerase in Prokaryotes: The core enzyme (α2ββ′ω)

and sigma factor (σ) make up the complete RNA polymerase
holoenzyme, which initiates transcription.
 3. Role of Sigma Factor (σ): The sigma subunit of RNA
polymerase recognizes and binds to the promoter, enabling the
RNA polymerase to locate the transcription start site.

4. Promoter Regions: The promoter contains conserved

sequences, particularly the -10 (TATAAT, also called the
Pribnow box) and -35 (TTGACA) regions, which are essential
for RNA polymerase binding.

5. Binding to Promoter: RNA polymerase holoenzyme binds to

the -35 and -10 sequences in the promoter, forming a closed
complex, where DNA remains double-stranded.

6. Formation of Open Complex: The DNA around the -10 region

unwinds, creating a transcription bubble of approximately 12-14
base pairs, forming the open complex.

7. Transcription Start Site: The first nucleotide (usually a purine,

like adenine or guanine) is selected based on the DNA template,
and RNA synthesis begins at the +1 site.

8. Abortive Initiation: Before stable RNA synthesis, RNA

polymerase may release short RNA fragments, known as
 abortive transcripts, multiple times before transitioning into
elongation.

9. Promoter Clearance: Once RNA polymerase synthesizes a

short RNA chain (usually around 10 nucleotides), it clears the
promoter and fully enters the elongation phase.

10. Role of NTPs: Ribonucleoside triphosphates (NTPs) are

added sequentially to the growing RNA strand, with the release
of pyrophosphate for each bond formed.

11. Sigma Factor Release: After promoter clearance, the sigma

factor is often released from the RNA polymerase complex,
allowing the core enzyme to proceed with elongation.

12. Efficiency of Initiation: The sigma factor aids in rapid

recognition and binding of RNA polymerase to promoters,
enhancing the efficiency and speed of transcription initiation.

13. Negative Regulation: Some genes have regulatory proteins

that can prevent RNA polymerase from binding to the promoter,
thus controlling transcription initiation.
 14. Importance of Initiation Phase: Proper initiation ensures
accurate and efficient transcription of genes, critical for cellular
function and response to environmental changes.

15. Biological Significance: Initiation regulation allows

prokaryotes to adapt rapidly to changing environments by
modulating gene expression, impacting metabolism, growth, and
survival.

3.(a) Write notes on Rho dependent transcription

termination. (5 Marks)

Rho-dependent termination is a mechanism in prokaryotes

where the rho protein facilitates termination of transcription,
ensuring that RNA synthesis stops accurately.

1. Definition: Rho-dependent termination is a process in which

the rho protein binds to specific sites on the nascent RNA,
causing the transcription complex to disassemble.

2. Structure of Rho: Rho is a hexameric ATPase protein that

binds RNA and utilizes ATP to translocate along the RNA.
3. Rho Binding Site: Rho binds to a region on the RNA known
as the rut (Rho utilization) site, which is rich in cytosine
residues.

4. Translocation of Rho: Using energy from ATP hydrolysis,

Rho moves along the RNA towards the RNA polymerase that is
synthesizing the transcript.

5. Helicase Activity: Rho’s helicase function unwinds the RNA-

DNA hybrid within the transcription complex, leading to the
release of the RNA molecule.

6. Termination Mechanism: Rho catches up to RNA polymerase

when it pauses at certain sites, leading to the disassembly of the
transcription complex.

7. Role in Gene Regulation: Rho-dependent termination

provides an additional layer of transcriptional regulation by
terminating certain transcripts selectively.
 8. Applications in Research: Understanding Rho-dependent
termination has implications in studying transcriptional
regulation and developing antibacterial agents.

9. Significance in Cellular Function: This termination

mechanism prevents unnecessary or overextended transcripts,
thus maintaining cellular efficiency.

10. Advantages and Limitations: While essential for

transcription regulation, it requires specific RNA sequences for
Rho binding, limiting its universality.

3.(b) Write notes on RNA polymerase. (5 Marks)

RNA polymerase is a central enzyme in transcription,

responsible for synthesizing RNA from a DNA template in
prokaryotes.

1. Function of RNA Polymerase: RNA polymerase catalyzes

RNA synthesis by adding ribonucleotides complementary to the
DNA template strand.
 2. Structure of Prokaryotic RNA Polymerase: It consists of a
core enzyme (α2ββ′ω) and a sigma factor (σ) that together form
the holoenzyme required for initiation.

3. Core Enzyme: The core enzyme carries out RNA synthesis

and elongation but cannot initiate transcription on its own.

4. Role of Sigma Factor: The sigma factor guides RNA

polymerase to specific promoter regions, initiating transcription.

5. Processivity: RNA polymerase is highly processive, capable

of synthesizing long RNA chains without dissociating from the
DNA template.

6. Elongation Function: During elongation, RNA polymerase

unwinds the DNA helix ahead and rewinds it behind as it
synthesizes RNA.

7. Proofreading: Prokaryotic RNA polymerase has limited

proofreading capabilities, sometimes backtracking to correct
errors.
8. Termination Role: RNA polymerase plays a role in both rho-
dependent and intrinsic termination, where it stops transcription
and releases the RNA.

9. Differences Across Domains: Prokaryotic RNA polymerases

differ structurally from eukaryotic RNA polymerases, which are
more complex and specialized.

10. Biological Significance: RNA polymerase is crucial for gene

expression, influencing cellular functions, growth, and response
to environmental cues.

3. Enumerating the four phases of transcription, describe the

initiation phase in detail. (10 Marks)

The transcription process in prokaryotes includes four phases:

initiation, elongation, termination, and recycling. The initiation
phase is critical, setting the stage for successful RNA synthesis.

1. Four Phases of Transcription: The phases include initiation,

elongation, termination, and recycling, each essential for
accurate RNA synthesis.
 2. Initiation Phase Overview: During initiation, RNA
polymerase binds to the DNA promoter, unwinds the DNA, and
begins RNA synthesis.

3. Role of RNA Polymerase Holoenzyme: RNA polymerase

holoenzyme, with the sigma factor, locates and binds to the
promoter.

4. Promoter Recognition: The -10 and -35 sequences on the

promoter are recognized by the sigma factor, guiding RNA
polymerase binding.

5. Closed Complex Formation: Upon binding, RNA polymerase

forms a closed complex with the double-stranded DNA.

6. Open Complex Formation: RNA polymerase unwinds the

DNA around the -10 region, creating an open complex and
exposing the template strand.

7. Transcription Start Site: RNA synthesis begins at the +1 site,

typically a purine nucleotide, on the DNA template strand.
8. Abortive Transcription: RNA polymerase may produce short
RNA sequences (abortive transcripts) until it successfully
transitions to elongation.

9. Sigma Factor Role: The sigma factor stabilizes the open

complex and facilitates the proper alignment of RNA
polymerase at the start site.

10. Initiation and Promoter Escape: Once the RNA chain reaches
a certain length (usually around 10 nucleotides), RNA
polymerase clears the promoter region.

11. Release of Sigma Factor: The sigma factor is often released

after promoter clearance, allowing the core enzyme to enter
elongation.

12. Regulatory Proteins: Transcription factors or repressors can

bind near the promoter to inhibit or enhance initiation,
controlling gene expression.

13. Biological Importance of Initiation: This phase determines

which genes are transcribed, crucial for the cell’s adaptability to
environmental changes.
14. Efficiency of Initiation: Initiation is a tightly regulated step,
as improper initiation can lead to errors in gene expression.

15. Examples in Prokaryotes: For instance, in E. coli, the lac

operon promoter regulates transcription initiation based on
lactose availability.

3.(a) Write a note on the conserved features of the promoter.

(5 Marks)

Promoters are specific DNA sequences that define the starting

point for transcription and determine the efficiency and
regulation of gene expression.

1. Definition: A promoter is a DNA sequence upstream of the

transcription start site, guiding RNA polymerase to initiate
transcription.

2. Core Promoter Elements: In prokaryotes, core promoter

elements include the -10 (Pribnow box) and -35 regions, critical
for RNA polymerase binding.
3. -10 Region (Pribnow Box): Located approximately 10 bases
upstream from the transcription start site, the Pribnow box
(TATAAT) is essential for forming the open complex by
facilitating DNA unwinding.

4. -35 Region: Situated around 35 bases upstream, the -35

sequence (TTGACA) provides a binding site for the sigma
factor, aiding in stable binding of RNA polymerase to the DNA.

5. Spacer Region: The spacer is the distance (typically 16-18

base pairs) between the -10 and -35 regions, which affects RNA
polymerase binding and transcription efficiency.

6. Consensus Sequence: Promoter sequences are highly

conserved across many genes, known as consensus sequences,
ensuring proper recognition by RNA polymerase.

7. UP Elements: Some promoters, especially in highly expressed

genes, contain UP elements upstream of the -35 region,
enhancing transcription by increasing RNA polymerase affinity.
 8. DNA Flexibility: Certain DNA sequences in the promoter
region provide structural flexibility, facilitating the unwinding
required for open complex formation.

9. Strength of Promoter: Promoters vary in “strength,” with

strong promoters allowing high transcription rates and weak
promoters causing lower transcription rates.

10. Biological Importance: Conserved promoter elements

regulate gene expression, essential for cellular functions and
response to environmental cues.

3.(b) Write a note on intrinsic transcription termination. (5

Marks)

Intrinsic (Rho-independent) termination is a mechanism in

prokaryotic transcription where the RNA sequence itself signals
the end of transcription.

1. Definition: Intrinsic termination is a process where

transcription terminates without the need for additional factors
like the rho protein.
2. Formation of Hairpin Structure: A GC-rich region in the RNA
forms a stable hairpin loop, followed by a series of uracil (U)
residues.

3. Role of Hairpin Loop: The hairpin structure causes physical

tension that disrupts the interaction between RNA polymerase
and the DNA template.

4. Poly-U Tail: Following the hairpin is a sequence of uracil

residues that weakens the RNA-DNA hybrid due to weak A-U
base pairs, aiding in termination.

5. Disassociation of RNA Polymerase: The combined effect of

the hairpin and poly-U tail destabilizes the transcription
complex, releasing RNA polymerase and the RNA transcript.

6. Energy Independence: Unlike rho-dependent termination,

intrinsic termination does not require ATP or other energy
sources.
 7. Widespread in Prokaryotes: This type of termination is
common in prokaryotes and occurs in genes with specific
sequences.

8. Efficiency: Intrinsic termination allows efficient and precise

transcription termination, preventing excessive or unintended
transcription.

9. Role in Gene Regulation: Termination at specific points

ensures proper gene expression and allows efficient use of
cellular resources.

10. Example: In E. coli, the trp operon terminates transcription

intrinsically when tryptophan levels are high, serving as a
regulatory mechanism.

3. Write short notes on:

(a) Transcription bubble. (5 Marks)

 The transcription bubble is an unwound section of DNA that
forms during transcription, allowing RNA polymerase to access
the template strand.

1. Definition: The transcription bubble is the region where DNA

unwinds to expose the template strand for RNA synthesis.

2. Formation: It forms as RNA polymerase separates the DNA

strands around the transcription start site, creating a single-
stranded template.

3. Length of Bubble: The transcription bubble typically spans

12-14 base pairs, providing enough space for RNA synthesis.

4. Role of RNA Polymerase: RNA polymerase unwinds the

DNA ahead of it and rewinds it behind, maintaining the
transcription bubble as it moves along the DNA.

5. Template Strand Exposure: The template strand within the

bubble is exposed for complementary base-pairing with
incoming ribonucleotides.
 6. Stability of the Bubble: The bubble remains stable during
elongation, with RNA polymerase shielding the single-stranded
DNA from nucleases.

7. Movement Along DNA: As RNA polymerase advances, the

bubble “travels” with it, unwinding ahead and rewinding behind.

8. Significance for Transcription: The transcription bubble

ensures accurate reading of the template strand and proper RNA
synthesis.

9. Comparison with Replication Bubble: Unlike replication

bubbles, transcription bubbles are smaller and transient, existing
only where RNA polymerase is actively transcribing.

10. Biological Importance: Maintaining the transcription bubble

is crucial for fidelity in gene expression, preventing DNA
damage and transcription errors.

3.(b) Elongation process of transcription. (5 Marks)

The elongation phase of transcription involves the synthesis of
RNA by RNA polymerase as it moves along the DNA template.

1. Definition: Transcription elongation is the phase where RNA

polymerase synthesizes RNA by adding nucleotides
complementary to the DNA template.

2. RNA Polymerase Function: RNA polymerase moves along

the DNA, unwinding and rewinding DNA, while synthesizing
RNA in the 5' to 3' direction.

3. Transcription Bubble Maintenance: RNA polymerase

maintains a transcription bubble, unwinding DNA ahead and
rewinding it behind as it progresses.

4. Nucleotide Addition: Ribonucleotides are added to the

growing RNA chain through complementary base pairing with
the DNA template.

5. Phosphodiester Bond Formation: Each nucleotide addition

involves the formation of a phosphodiester bond, releasing
pyrophosphate and elongating the RNA.
 6. Backtracking and Proofreading: RNA polymerase can
backtrack to correct errors, enhancing transcription fidelity.

7. Speed of Elongation: In E. coli, elongation occurs at

approximately 40-50 nucleotides per second, though this rate
may vary.

8. Transcription Pausing: RNA polymerase may pause at

specific sites, allowing for regulatory events, folding of RNA, or
error correction.

9. Interactions with Elongation Factors: Elongation factors may

assist in preventing RNA polymerase from stalling, facilitating
smooth transcription.

10. Biological Importance: The elongation phase is essential for

producing functional RNA molecules, with each RNA product
impacting cellular activities.

Certainly! Here are the answers to your questions in the required

format:
 4. Write notes on:

(a) Reverse Transcription. (5 Marks)

Reverse transcription is a process in which RNA is converted

back into DNA. This mechanism is utilized by retroviruses to
integrate their genetic material into the host genome, allowing
the virus to replicate within the host cell.

1. Definition: Reverse transcription is the synthesis of DNA

from an RNA template, performed by the enzyme reverse
transcriptase.

2. Enzyme Involved: Reverse transcriptase is the enzyme

responsible for converting single-stranded RNA into
complementary DNA (cDNA).

3. Process Overview: The RNA template is copied into DNA,

followed by the synthesis of a second DNA strand to form
double-stranded DNA.

4. Importance in Retroviruses: Retroviruses, such as HIV, use

reverse transcription to integrate their RNA genome into the
 DNA of the host cell, allowing them to hijack the host’s
replication machinery.

5. Formation of Provirus: The double-stranded DNA produced

integrates into the host genome, forming a provirus that can be
transcribed and translated by the host.

6. Applications in Biotechnology: Reverse transcription is

crucial in techniques like RT-PCR, where RNA is converted into
DNA for amplification and analysis.

7. Evolutionary Significance: The presence of reverse

transcriptase in viruses suggests an evolutionary relationship, as
reverse transcription is thought to have played a role in the
development of retroelements.

8. Medical Implications: Reverse transcription is a target for

antiviral drugs, like reverse transcriptase inhibitors, which are
used in treating HIV.

9. Limitations: The process has a high error rate, which can lead
to mutations, contributing to the rapid evolution of retroviruses.
10. Examples: Besides HIV, other retroviruses, including HTLV
(Human T-lymphotropic virus), also utilize reverse transcription
for infection and replication.

4. Give a detailed account of the structure and working of

the lac operon. (10 Marks)

The lac operon is a well-studied operon system in E. coli that

enables the bacteria to utilize lactose as a carbon source. It
provides an example of gene regulation in prokaryotes and is
controlled by both positive and negative mechanisms.

1. Definition: The lac operon is a cluster of genes that code for

enzymes involved in lactose metabolism, regulated by a single
promoter.

2. Gene Components: It includes three structural genes - lacZ,

lacY, and lacA - encoding β-galactosidase, lactose permease, and
thiogalactoside transacetylase, respectively.
3. LacZ (β-Galactosidase): This enzyme hydrolyzes lactose into
glucose and galactose, allowing the bacteria to utilize lactose as
an energy source.

4. LacY (Lactose Permease): Lactose permease facilitates the

transport of lactose into the bacterial cell.

5. LacA (Transacetylase): The function of thiogalactoside

transacetylase is less understood but may play a role in
detoxifying by-products of lactose metabolism.

6. Promoter (P) and Operator (O): The promoter is where RNA

polymerase binds to initiate transcription, while the operator is a
regulatory sequence where the lac repressor binds.

7. Regulation by Lac Repressor (LacI): In the absence of lactose,

the LacI protein binds to the operator, blocking RNA
polymerase from transcribing the operon.

8. Induction by Lactose: When lactose is present, it binds to the

lac repressor, causing a conformational change that prevents the
repressor from binding to the operator.
9. Role of Allolactose: Allolactose, a lactose metabolite, acts as
an inducer that binds to the repressor, inactivating it and
allowing transcription.

10. Catabolite Activator Protein (CAP): When glucose is low,

CAP binds to cAMP, and this complex enhances RNA
polymerase binding, increasing transcription.

11. Role of cAMP: cAMP levels rise when glucose is scarce,

binding with CAP to stimulate transcription and ensure lactose
utilization.

12. Negative Control: The lac repressor is an example of

negative control, preventing transcription when lactose is absent.

13. Positive Control by CAP: The cAMP-CAP complex

provides positive control by enhancing transcription in low
glucose conditions.

14. Significance of Dual Control: The lac operon is controlled

both by repression and activation, allowing efficient and
economical gene expression based on nutrient availability.
 15. Biological Importance: The lac operon demonstrates the
adaptability of E. coli, enabling it to switch between different
energy sources and conserve resources.

4.(a) Give mechanism of attenuation in 'trp' operon. (5

Marks)

Attenuation is a regulatory mechanism in the trp operon that

allows transcription to respond to tryptophan levels in the cell by
terminating transcription prematurely when tryptophan is
abundant.

1. Definition: Attenuation is a regulatory process that controls

the termination of transcription based on the intracellular
concentration of tryptophan.

2. Location: The attenuation mechanism operates in the leader

sequence of the trp operon, located between the promoter and
structural genes.
3. Leader Peptide: The leader sequence encodes a short peptide
containing tryptophan residues, which plays a role in sensing
tryptophan levels.

4. Ribosome Stalling: When tryptophan is low, the ribosome

stalls while translating the leader peptide due to a shortage of
charged tRNA for tryptophan.

5. Formation of Anti-Terminator: Ribosome stalling causes an

anti-terminator structure to form in the mRNA, allowing
transcription to continue through the structural genes.

6. Terminator Hairpin: When tryptophan is abundant, the

ribosome does not stall, and a terminator hairpin forms, halting
transcription prematurely.

7. Tryptophan Abundance: High tryptophan levels result in a

terminator loop formation, preventing transcription of the
downstream genes.

8. Fine-Tuning Expression: Attenuation allows the cell to fine-

tune the expression of tryptophan biosynthetic genes according
to need.
9. Energy Conservation: This mechanism prevents unnecessary
synthesis of enzymes for tryptophan synthesis, saving cellular
energy.

10. Example in E. coli: The trp operon in E. coli uses attenuation

as a secondary control mechanism to respond to tryptophan
availability.

4.(b) Discuss reverse transcription in retrovirus. (5 Marks)

Retroviruses use reverse transcription to convert their RNA

genome into DNA, which integrates into the host genome and
enables the virus to replicate within host cells.

1. Definition: In retroviruses, reverse transcription is the

conversion of RNA into DNA, allowing viral genetic material to
integrate into the host genome.

2. Retroviral RNA Genome: Retroviruses, like HIV, carry an

RNA genome that must be reverse-transcribed into DNA to
integrate into the host DNA.
3. Enzyme - Reverse Transcriptase: Retroviruses contain reverse
transcriptase, which catalyzes the synthesis of complementary
DNA (cDNA) from the viral RNA.

4. Two-Step Synthesis: Reverse transcription involves creating a

single-stranded DNA from RNA, followed by synthesis of a
complementary DNA strand, forming double-stranded DNA.

5. Integration into Host Genome: The double-stranded DNA

integrates into the host cell’s genome, becoming a permanent
part of the host's genetic material.

6. Formation of Provirus: Once integrated, the viral DNA is

referred to as a provirus and can be transcribed and translated by
the host’s cellular machinery.

7. Role in Viral Replication: The provirus directs the synthesis

of new viral RNA and proteins, which assemble into new virus
particles.
 8. High Mutation Rate: Reverse transcription lacks
proofreading, leading to frequent mutations that contribute to the
evolution and drug resistance of retroviruses.

9. Medical Significance: Understanding reverse transcription is

essential in developing antiretroviral therapies, as inhibitors of
reverse transcriptase are effective against HIV.

10. Example - HIV: HIV uses reverse transcription to convert its

RNA genome into DNA, allowing it to establish a chronic
infection in host cells.

4. (a) Giving the structure of trp operon, discuss its negative

control. (5 Marks)

The trp operon is a repressible operon in E. coli that is regulated

by negative feedback control based on tryptophan availability,
which allows it to be turned off when tryptophan levels are high.

1. Definition: The trp operon is a group of genes responsible for

synthesizing tryptophan, regulated by negative feedback to
prevent excess production.
 2. Gene Components: It includes five structural genes (trpE,
trpD, trpC, trpB, and trpA), each encoding enzymes for
tryptophan biosynthesis.

3. Regulatory Elements: The operon has a promoter and an

operator region upstream of the structural genes, which are sites
for regulatory protein binding.

4. Role of Tryptophan as Corepressor: Tryptophan itself acts as a

corepressor, binding to the trp repressor protein to activate it.

5. Trp Repressor Protein: The trp repressor, when activated by

tryptophan, binds to the operator, blocking RNA polymerase
from initiating transcription.

6. Mechanism of Negative Control: When tryptophan is

abundant, it binds to the repressor, enabling it to inhibit
transcription of the trp operon.

7. Prevention of Wasteful Synthesis: This regulation prevents the

unnecessary production of enzymes when tryptophan is already
available, conserving energy.
 8. Feedback Inhibition: This negative control mechanism is a
form of feedback inhibition, where the end product (tryptophan)
regulates its own synthesis.

9. Relief of Repression: When tryptophan levels fall, the

repressor dissociates from the operator, allowing transcription
and tryptophan synthesis to resume.

10. Example in Prokaryotes: The trp operon in E. coli is a classic

example of a repressible operon regulated by end-product
inhibition.

4. (a) Explain the role of cAMP in the lac operon. (5 Marks)

cAMP (cyclic adenosine monophosphate) is a signaling

molecule that regulates the lac operon by enhancing the binding
of RNA polymerase, promoting efficient transcription when
glucose is low.
 1. Definition: cAMP is a secondary messenger that modulates
gene expression in response to nutrient availability, particularly
glucose.

2. Connection to Glucose Levels: When glucose is scarce,

cAMP levels rise in E. coli, signaling the need for alternative
energy sources like lactose.

3. cAMP-CRP Complex: cAMP binds to the catabolite activator

protein (CRP), forming the cAMP-CRP complex.

4. Enhancing RNA Polymerase Binding: The cAMP-CRP

complex binds to the promoter region of the lac operon,
increasing RNA polymerase’s affinity for the promoter.

5. Positive Control Mechanism: This binding acts as a positive

control, facilitating transcription of the lac operon genes when
lactose is available and glucose is not.

6. Transcriptional Activation: In the presence of lactose and

absence of glucose, the cAMP-CRP complex allows high levels
of transcription, maximizing lactose metabolism.
 7. Allosteric Regulation: cAMP binding to CRP induces a
conformational change, allowing CRP to bind to the DNA and
activate transcription.

8. Catabolite Repression: In high glucose conditions, cAMP

levels are low, and the lack of cAMP-CRP complex results in
reduced lac operon transcription (catabolite repression).

9. Energy Conservation: This regulation ensures that E. coli uses

the most efficient energy source first, conserving resources.

10. Example in E. coli: The role of cAMP in regulating the lac

operon in E. coli exemplifies how cells adapt to nutrient
availability through gene regulation.

4.Write notes on:

(a) Enzymes encoded by lac operon and their role. (2.5

Marks)
 The lac operon encodes enzymes essential for lactose
metabolism, allowing E. coli to utilize lactose as an energy
source when glucose is unavailable.

1. β-Galactosidase (lacZ): Converts lactose into glucose and

galactose by hydrolyzing the β-glycosidic bond.

2. Lactose Permease (lacY): A membrane protein that transports

lactose into the bacterial cell.

3. Transacetylase (lacA): Catalyzes the transfer of an acetyl

group to thiogalactosides, though its role in lactose metabolism
is not fully understood.

4. Role in Lactose Utilization: Together, these enzymes enable

the cell to metabolize lactose when it is present.

5. Induction by Lactose: Expression of these enzymes is induced

by lactose, allowing efficient resource allocation.

6. Energy Production: β-Galactosidase’s activity provides

glucose, an energy source for cellular metabolism.
7. Adaptability of E. coli: The presence of these enzymes allows
E. coli to adapt to environments where lactose is the primary
carbon source.

8. Example in Metabolism: This enzyme set illustrates how

bacteria adjust gene expression to match available nutrients.

(b) What is attenuation? (2.5 Marks)

Attenuation is a regulatory mechanism that fine-tunes gene

expression by prematurely terminating transcription, based on
metabolite levels.

1. Definition: Attenuation is a gene regulatory mechanism in

prokaryotes that uses premature transcription termination based
on metabolite availability.

2. Example - trp Operon: In the trp operon, attenuation responds

to tryptophan levels, halting transcription when tryptophan is
abundant.
 3. Leader Sequence: The attenuation mechanism involves a
leader sequence that encodes a short peptide, helping to sense
metabolite levels.

4. Termination Mechanism: When the metabolite is abundant, a

terminator structure forms in the mRNA, causing transcription
to stop.

5. Ribosome Pausing: In the trp operon, ribosome stalling at the

leader peptide occurs when tryptophan is scarce, allowing
transcription to continue.

6. Control of Biosynthetic Pathways: Attenuation regulates

biosynthetic operons, conserving resources by halting
unnecessary synthesis.

7. Rapid Response to Changes: Attenuation allows cells to

quickly adjust gene expression based on metabolite availability.

8. Significance in E. coli: Attenuation in the trp operon is an

example of how cells use transcriptional control to optimize
energy usage.
(c) Rifampicin as inhibitor of prokaryotic transcription. (2.5
Marks)

Rifampicin is an antibiotic that inhibits bacterial transcription by

targeting RNA polymerase, preventing RNA synthesis.

1. Mechanism of Action: Rifampicin binds to the β subunit of

bacterial RNA polymerase, blocking RNA chain elongation.

2. Selective Targeting: The drug specifically targets bacterial

RNA polymerase, making it effective against bacteria without
affecting human RNA polymerase.

3. Usage in TB Treatment: Rifampicin is widely used to treat

tuberculosis (TB) due to its efficacy against Mycobacterium
tuberculosis.

4. Prevention of mRNA Synthesis: By blocking RNA synthesis,

rifampicin prevents bacteria from producing essential proteins,
leading to cell death.
 5. Resistance Development: Bacterial mutations in RNA
polymerase can confer rifampicin resistance, a major challenge
in TB treatment.

6. Side Effects: Long-term use of rifampicin may cause liver

damage, and it is known to induce hepatic enzymes.

7. Combination Therapy: To avoid resistance, rifampicin is often

combined with other antibiotics in TB therapy.

8. Importance in Research: Rifampicin is also used in laboratory

studies to investigate transcriptional mechanisms.

(d) Negative control of trp operon. (2.5 Marks)

The trp operon is regulated by negative control, allowing

transcription to be repressed when tryptophan levels are high,
conserving cellular resources.
 1. Definition: Negative control involves repressing gene
transcription when the end product (tryptophan) is abundant.

2. Trp Repressor Protein: The trp repressor binds to the operator

region of the operon in the presence of tryptophan.

3. Corepressor Role of Tryptophan: Tryptophan acts as a

corepressor, activating the trp repressor to inhibit transcription.

4. Operator Binding: The activated trp repressor binds to the

operator, blocking RNA polymerase from initiating
transcription.

5. Energy Conservation: This regulation prevents the cell from

expending resources on tryptophan synthesis when it is not
needed.

6. Feedback Inhibition: The process is a classic example of

feedback inhibition, where the product inhibits its own
synthesis.

7. Repression Relief: When tryptophan levels are low, the

repressor cannot bind
to the operator, allowing transcription to proceed and tryptophan
biosynthesis to resume.

8. Example in E. coli: The negative control of the trp operon in

E. coli serves as an example of efficient resource management in
response to nutrient availability.

4. Describe in detail the attenuation mechanism of ‘trp’

operon. (10 Marks)

The attenuation mechanism of the trp operon in E. coli provides

an additional layer of regulation by adjusting transcription based
on intracellular tryptophan levels, allowing fine-tuned control
over gene expression.

1. Definition of Attenuation: Attenuation is a regulatory

mechanism that causes premature termination of transcription,
allowing cells to modulate gene expression based on metabolite
levels, such as tryptophan.
 2. Role in trp Operon: In the trp operon, attenuation
complements the repressor mechanism, further controlling
tryptophan biosynthesis when levels of tryptophan are high.

3. Leader Sequence: The trp operon has a 162-base pair leader

sequence, located upstream of the structural genes, which plays
a central role in the attenuation process.

4. Leader Peptide Coding: This leader sequence contains a short

open reading frame that encodes a leader peptide with two
adjacent tryptophan codons, making it sensitive to tryptophan
availability.

5. Formation of Secondary Structures: The mRNA produced

from the leader sequence can form different stem-loop structures
(hairpins), depending on ribosome position, which affect
transcription continuation or termination.

6. Attenuator Region: Just downstream of the leader peptide

sequence, there is an attenuator region that can fold into
terminator or anti-terminator structures based on tryptophan
levels.
 7. High Tryptophan Levels - Terminator Formation: When
tryptophan is abundant, ribosomes quickly translate the leader
peptide, allowing a terminator hairpin (a 3-4 stem-loop) to form
in the mRNA. This structure halts transcription.

8. Low Tryptophan Levels - Anti-terminator Formation: When

tryptophan levels are low, the ribosome stalls at the tryptophan
codons in the leader sequence, allowing an anti-terminator
structure (2-3 stem-loop) to form, which permits transcription to
continue.

9. Sensitivity to Tryptophan Availability: This stalling

mechanism directly links ribosome progression to tryptophan
levels, making the attenuation mechanism highly responsive to
metabolic conditions.

10. Prevention of Wasteful Synthesis: By halting transcription

when tryptophan is abundant, attenuation prevents unnecessary
enzyme production, conserving cellular energy.

11. Comparison with Repressor Mechanism: While the trp

repressor stops transcription initiation in the presence of
tryptophan, attenuation provides a secondary control during
transcription elongation.
 12. Dynamic Control of Transcription: The two-tiered control
(repressor and attenuation) allows the cell to adjust gene
expression dynamically as tryptophan levels fluctuate.

13. Role in Other Amino Acid Operons: Attenuation is a

regulatory strategy used by other amino acid biosynthesis
operons, such as histidine and phenylalanine operons in
prokaryotes.

14. Importance in Prokaryotic Regulation: Attenuation

exemplifies prokaryotic regulatory strategies that allow bacteria
to adapt rapidly to changes in nutrient availability.

15. Example of Feedback Regulation: The attenuation

mechanism in the trp operon illustrates feedback regulation,
where gene expression is modulated in response to intracellular
concentrations of a metabolite, in this case, tryptophan.

Here are the answers to each question, provided in numerical

order and concisely answered for 1 mark, with each question
stated before its answer.
 1 MARKS QUE :-

Similar Questions (Repetitive Concepts)

1. What is lagging strand of DNA replication?

The lagging strand is synthesized discontinuously in short

Okazaki fragments in the 5' to 3' direction, opposite to the
replication fork movement.

2. Explain the term 'lagging strand'.

The lagging strand is the DNA strand synthesized in fragments

during replication, as DNA polymerase works in the 5' to 3'
direction.

3. What are leading and lagging strands?

The leading strand is synthesized continuously, while the

lagging strand is synthesized discontinuously in short fragments
called Okazaki fragments.
 4. What is Klenow fragment?

The Klenow fragment is a portion of DNA polymerase I with

polymerase and 3' to 5' exonuclease activities but lacks 5' to 3'
exonuclease activity.

5. Define role of sigma factor.

The sigma factor helps RNA polymerase recognize and bind to

specific promoter regions, initiating transcription.

6. Give the role of sigma subunit.

The sigma subunit directs RNA polymerase to specific promoter

regions on DNA for the start of transcription.

7. What is the role of sigma factor?

The sigma factor enables RNA polymerase to locate and bind to

promoter sequences, initiating transcription.
 8. What is TATA box?

The TATA box is a promoter sequence found in eukaryotic DNA

that helps recruit transcription factors and RNA polymerase

9. What is Pribnow box?

The Pribnow box is a prokaryotic promoter sequence (similar to

TATA box) that helps initiate transcription.

10. Give the consensus sequence of Pribnow box.

The consensus sequence of the Pribnow box is TATAAT, located

about 10 bases upstream of the transcription start site.

11. Name the inhibitors of prokaryotic transcription.

Rifampicin is an example of a prokaryotic transcription

inhibitor.

12. Name types of DNA polymerases in prokaryotes.

 DNA polymerases I, II, and III are the main types in
prokaryotes.

13. What are types of DNA polymerases?

Prokaryotes primarily use DNA polymerases I, II, and III for

DNA synthesis and repair.

14. What do you mean by the term 'attenuation'?

Attenuation is a regulatory mechanism in operons where

transcription is prematurely terminated based on metabolite
levels.

15. What are Okazaki fragments?

Okazaki fragments are short DNA segments synthesized on the

lagging strand during DNA replication.

16. What is inducer in an operon?

An inducer is a molecule that activates gene expression, like
allolactose in the lac operon.

17. Role of allolactose in lactose operon.

Allolactose binds to the lac repressor, causing it to release the

DNA and allowing transcription to proceed.

18. What is meant by 'priming' in DNA replication?

Priming involves creating a short RNA primer to initiate DNA

synthesis on the template strand.

19. What is meant by 'Operon'?

An operon is a group of genes regulated together by a single

promoter and regulatory sequence.

Unique Questions
 1. What is primosome?

The primosome is a protein complex responsible for initiating

DNA replication by synthesizing RNA primers.

2. What is the role of helicase enzyme?

Helicase unwinds the DNA double helix at the replication fork,

creating single-stranded templates.

3. What is base analog?

A base analog is a compound similar to DNA bases that can

incorporate into DNA, sometimes causing mutations.

4. What is C-value?

The C-value is the amount of DNA in a haploid genome of an

organism, measured in base pairs or picograms.
 5. What is TATA box?

The TATA box is a DNA sequence in eukaryotic promoters that

binds transcription factors, aiding transcription initiation.

6. What are downstream sequences?

Downstream sequences are DNA regions located in the 3'

direction after the transcription start site.

7. Full form of IPTG.

IPTG stands for Isopropyl β-D-1-thiogalactopyranoside.

8. Which enzyme is responsible for the synthesis of

allolactose?

β-galactosidase catalyzes the synthesis of allolactose from

lactose.

9. What is nick translation?

 Nick translation is a DNA repair process where DNA
polymerase I removes nucleotides ahead of a nick and replaces
them with new nucleotides.

10. Name the technique for determination of length of

promoter.

Promoter length is often determined using reporter gene assays

or DNA footprinting techniques.

11. What enzyme activity is responsible for replication

fidelity?

The 3' to 5' exonuclease activity (proofreading) of DNA

polymerase ensures replication fidelity.

12. Give subunit composition of prokaryotic RNA

polymerase.

The core RNA polymerase in prokaryotes consists of α₂, β, β',

and ω subunits, with a sigma (σ) factor for initiation.
 13. What is the role of -35 sequences in transcription?

The -35 sequence in prokaryotic promoters binds the sigma

factor of RNA polymerase, aiding transcription initiation.

14. Name the enzyme which cleaves β, 1-4 linkage of lactose.

β-galactosidase cleaves the β, 1-4 linkage in lactose.

15. Give example of mutation caused by UV in DNA.

Thymine dimers are a common mutation caused by UV

exposure in DNA.

16. Why mismatch repair is also called as mut HLS System?

Mismatch repair involves MutH, MutL, and MutS proteins,

hence it’s also called the MutHLS system.

17. Which DNA polymerase is responsible for SOS repair?

DNA polymerase V is primarily responsible for SOS repair in
prokaryotes.