Transcription (RNA Synthesis)

E-CONTENT

TRANSCRIPTION

The Molecular Basis of RNA Synthesis

UGC Four Quadrant Approach

Course: B.Sc. Molecular Biology

Level: Undergraduate (B.Sc. 6th Sem)

Prepared by Dr Chandralekha Deka

Assistant Professor

Department of Zoology

PDUAM, Amjonga, Goalpara

QUADRANT I e-Tutorial (Main Content)

1.1 Introduction to Transcription

1.2 RNA Polymerase Structure

1.3 Transcriptional Unit

1.4 Mechanism of Transcription – Prokaryotes

1.5 Mechanism of Transcription – Eukaryotes

1.6 Post-Transcriptional Modifications

QUADRANT II e-Content (Supplementary Material)

2.1 Key Definitions

2.2 Comparison Tables

2.3 Summary Notes

QUADRANT III Self-Assessment

3.1 Multiple Choice Questions (MCQs)

3.2 Short Answer Questions

3.3 Long Descriptive Questions

3.4 Assertion-Reason Questions

QUADRANT IV Additional Resources

4.1 Textbook References

4.2 Journal Articles (APA)

4.3 Digital Learning Resources

QUADRANT I

e-Tutorial | Main Content

1. Introduction to Transcription

1.1 Definition and Significance

Transcription is the biological process by which genetic information encoded in DNA is copied into a complementary RNA molecule. This process is catalysed by the enzyme RNA polymerase and represents the first major step in the flow of genetic information from genotype to phenotype.

KEY DEFINITION: Transcription is defined as the enzymatic synthesis of an RNA molecule using a DNA template, where the nucleotide sequence of the RNA is complementary to one strand of the DNA double helix.

Transcription is significant because:

• It converts the stable, double-stranded information in DNA into single-stranded RNA molecules that can be translated into proteins.

• It is the primary step at which gene expression is regulated in all organisms.

• It produces multiple RNA copies from a single gene, thereby amplifying the genetic signal.

• Different types of RNA (mRNA, tRNA, rRNA) produced by transcription serve different roles in protein synthesis.

• Errors or dysregulation in transcription are associated with diseases including cancer and metabolic disorders.

1.2 Central Dogma of Molecular Biology

The Central Dogma, first proposed by Francis Crick in 1958, describes the directional flow of genetic information within a biological system:

DNA → (Replication) → DNA

DNA → (Transcription) → RNA

RNA → (Translation) → Protein

The 'special flows' described later by Crick include reverse transcription (RNA to DNA, as in retroviruses) and direct RNA replication (RNA to RNA, as in RNA viruses). However, the core dogma remains applicable to all cellular life.

1.3 Overview of RNA Synthesis

RNA synthesis (transcription) involves the following general steps:

1. Template Recognition: RNA polymerase binds to a specific DNA sequence called the promoter.

2. Strand Separation: The DNA double helix is locally unwound to expose the template strand.

3. Phosphodiester Bond Formation: RNA polymerase synthesises RNA in the 5' to 3' direction, adding ribonucleotides complementary to the template strand (reading 3' to 5').

4. Elongation: The RNA chain grows as the polymerase moves along the template.

5. Termination: Synthesis stops at a specific terminator sequence; the RNA transcript is released.

NOTE: Unlike DNA replication, transcription does NOT require a primer. RNA polymerase can initiate new chains de novo. Also, only a small portion of the DNA is transcribed at any given time.

2. RNA Polymerase Structure

2.1 Prokaryotic RNA Polymerase

Bacteria possess a single, multi-subunit RNA polymerase (RNAP) capable of transcribing all classes of RNA genes. The complete functional enzyme is called the holoenzyme.

Core Enzyme Subunit Composition

Subunit	Function
2 × Alpha (α)	Assembly platform; interacts with regulatory factors; each ~36 kDa
Beta (β)	Contains the catalytic centre; binds incoming NTPs; ~150 kDa
Beta-prime (β')	Binds DNA template non-specifically; ~155 kDa
Omega (ω)	Assists assembly and stability of β'; ~10 kDa

The core enzyme composition is written as: α₂ββ'ω

The holoenzyme includes an additional dissociable subunit called the sigma factor (σ): α₂ββ'ωσ

Role of the Sigma Factor

• The sigma (σ) factor confers promoter specificity to the RNA polymerase.

• Without σ, the core enzyme binds DNA non-specifically and weakly.

• With σ, the holoenzyme recognises and tightly binds specific promoter sequences.

• After initiation, σ dissociates and is recycled for use by other core enzymes.

• E. coli has multiple sigma factors: σ70 (housekeeping genes), σ32 (heat shock), σ54 (nitrogen metabolism), etc.

2.2 Eukaryotic RNA Polymerases

Eukaryotes have three major nuclear RNA polymerases, each transcribing a specific class of genes:

RNA Polymerase	Location	Transcribes
RNA Pol I	Nucleolus	Large rRNA precursor (45S → 28S, 18S, 5.8S rRNA)
RNA Pol II	Nucleoplasm	mRNA precursors (hnRNA), most snRNA
RNA Pol III	Nucleoplasm	tRNA, 5S rRNA, small RNA (7SL, U6 snRNA)

Additionally, plants possess RNA Pol IV and Pol V involved in RNA-directed DNA methylation (gene silencing). Mitochondria and chloroplasts have their own distinct single-subunit RNA polymerases resembling bacteriophage T7 polymerase.

EXAM TIP: RNA Pol II is the most important in eukaryotes because it synthesises mRNA, which encodes proteins. It is the target of the toxin alpha-amanitin (from Amanita phalloides) at very low concentrations.

2.3 Functional Differences: Prokaryotic vs Eukaryotic RNAP

Feature	Prokaryotes	Eukaryotes
Number of RNAPs	One (for all RNA)	Three major (Pol I, II, III)
Complexity	~400 kDa (5 subunits)	~500–700 kDa (12–15 subunits)
Promoter recognition	Via sigma factor	Via general transcription factors (GTFs)
Coupling with translation	Yes (simultaneous)	No (nucleus vs cytoplasm)
Inhibition	Rifampicin (binds β)	α-amanitin (binds Pol II CTD)
RNA processing	Minimal	Extensive (capping, splicing, poly-A)

3. Transcriptional Unit

3.1 Definition

A transcriptional unit is the region of DNA that is transcribed into a single RNA molecule. It encompasses all the sequences required for the initiation, elongation, and termination of transcription.

3.2 Components

Promoter

• A promoter is a cis-acting DNA sequence located upstream (5') of the structural gene to which RNA polymerase binds to initiate transcription.

• In prokaryotes, the consensus sequences at -10 (Pribnow box: TATAAT) and -35 (TTGACA) are critical promoter elements.

• In eukaryotes, core promoter elements include the TATA box (TATAAA, ~-25), Initiator element (Inr), and downstream promoter element (DPE).

Structural Gene (Coding Region)

• The structural gene contains the DNA sequence that is transcribed into RNA.

• It begins at the transcription start site (+1) and ends at the terminator.

• In prokaryotes, structural genes for functionally related proteins are often organised into operons.

Terminator

• A DNA sequence that signals the end of transcription.

• In prokaryotes: either a GC-rich palindrome followed by a poly-T run (intrinsic/Rho-independent) or a Rho-dependent mechanism.

• In eukaryotes: termination is less well-defined and coupled with polyadenylation signals.

3.3 Template Strand vs Coding Strand

Template Strand (Antisense)	Coding Strand (Sense / Non-template)
Read by RNA polymerase (3'→5' direction)	Same sequence as the RNA (5'→3')
Acts as the blueprint for RNA synthesis	Also called the sense or non-template strand
Also called antisense or minus strand	Sequence used to write the gene in textbooks
RNA produced is complementary to this strand	Contains the same codons as the mRNA (with U instead of T)

MEMORY AID: The Coding Strand has the same sequence as the mRNA (with T instead of U). The Template Strand is the one actually READ by RNA polymerase.

3.4 Regulatory Sequences

Beyond the core promoter, transcription is regulated by additional DNA elements:

• Enhancers: DNA sequences that increase transcription; can act at great distances (up to 1 Mb) and in any orientation.

• Silencers: Sequences that repress transcription; bind repressor proteins.

• Insulators/Boundary elements: Separate active from inactive chromatin domains.

• Response Elements: Sequences that bind specific transcription factors activated by signals (e.g., CRE for cAMP response; HRE for hormones).

• Upstream Activating Sequences (UAS): Yeast regulatory elements analogous to enhancers.

4. Mechanism of Transcription in Prokaryotes

4.1 Stage 1: Initiation

Initiation is the most highly regulated phase of transcription and occurs through a series of well-defined steps:

· The sigma (σ) factor binds to the core RNA polymerase enzyme, forming the holoenzyme, which is capable of recognizing promoter sequences.

· The holoenzyme attaches to the promoter region of DNA, forming a closed complex, where the DNA remains double-stranded.

· The enzyme then unwinds approximately 17 base pairs of DNA near the –10 region (Pribnow box), creating an open complex or transcription bubble.

· The first two ribonucleoside triphosphates (NTPs) align on the template strand and are joined to form a dinucleotide, marking the beginning of RNA synthesis.

· Several short RNA fragments may be produced and released during abortive initiation, before stable elongation begins.

· Once the RNA polymerase successfully moves away from the promoter (promoter clearance), the sigma factor dissociates.

· The remaining core enzyme continues the process of RNA chain elongation.

This step ensures accurate initiation of transcription and plays a crucial role in gene regulation.

The E. coli sigma factor σ70 recognises the -10 (TATAAT) and -35 (TTGACA) promoter elements. The spacer between -10 and -35 is typically 17 ± 1 bp. Mutations in these elements markedly reduce transcription efficiency.

4.2 Stage 2: Elongation

Elongation is the phase of transcription in which RNA polymerase travels along the DNA template, synthesizing a complementary RNA strand through the sequential addition of nucleotides. After clearing the promoter, the enzyme enters this active phase of RNA synthesis. In E. coli, transcription proceeds at a rate of about 40–50 nucleotides per second.

During this process, a transcription bubble of approximately 17 base pairs is maintained, within which an RNA–DNA hybrid of about 8–9 base pairs is formed. As RNA polymerase advances in the 5′ → 3′ direction, the DNA ahead of the enzyme is unwound, while the DNA behind it re-anneals.

The movement of the polymerase generates torsional strain in the DNA, which is relieved by topoisomerases (Topoisomerase I and Topoisomerase II/DNA gyrase). Additionally, elongation factors such as NusA (which enhances pausing) and NusG (which suppresses pausing) play important roles in regulating the speed and efficiency of transcription.

4.3 Stage 3: Termination

A. Rho-Independent (Intrinsic) Termination

· This type of termination occurs without the involvement of any protein factor and is therefore called intrinsic termination.

· A GC-rich palindromic sequence in the newly synthesized RNA folds into a stem–loop (hairpin) structure, followed by a stretch of 6–8 uridine (U) residues.

· The formation of the hairpin structure causes RNA polymerase to pause and weakens the stability of the RNA–DNA hybrid.

· Additionally, the presence of weak rU–dA base pairs further destabilizes the hybrid, leading to the release of the RNA transcript.

B. Rho-Dependent Termination

· This mechanism requires the Rho protein, an ATP-dependent RNA helicase (homohexamer, ~300 kDa).

· Rho binds to a specific sequence on the RNA called the Rho utilization (rut) site, which is typically unstructured and rich in cytosine (C) residues.

· It then moves along the RNA in the 5′ → 3′ direction, utilizing energy from ATP hydrolysis.

· When RNA polymerase pauses at a termination site, Rho catches up to the enzyme and unwinds the RNA–DNA hybrid, resulting in the release of the transcript.

· Nearly 50% of transcription termination events in E. coli occur through the Rho-dependent mechanism.

Rho-Independent Termination	Rho-Dependent Termination
No protein factor required	Requires Rho protein (hexameric helicase)
GC-rich hairpin + poly-U tail	Rho loads at rut site (C-rich, unstructured)
Hairpin stalls RNAP	Rho translocates, unwinds RNA-DNA hybrid
~50% of E. coli terminators	~50% of E. coli terminators
Also called intrinsic termination	Also called factor-dependent termination

5. Mechanism of Transcription in Eukaryotes

Eukaryotic transcription is considerably more complex than prokaryotic transcription. Here we focus primarily on transcription by RNA Polymerase II (mRNA synthesis).

5.1 Initiation

Eukaryotic RNA Pol II cannot bind the promoter directly. It requires General Transcription Factors (GTFs) that assemble at the core promoter to form the Pre-Initiation Complex (PIC).

Core Promoter Elements

• TATA Box (Hogness Box): Consensus TATAAA, located ~25-30 bp upstream of the TSS; present in ~24% of genes.

• Initiator Element (Inr): Spans the TSS (+1); consensus PyPyAN(T/A)PyPy.

• Downstream Promoter Element (DPE): Located ~30 bp downstream of TSS; compensates when TATA box is absent.

• TFIIB Recognition Element (BRE): Flanks the TATA box; binds TFIIB.

Assembly of the Pre-Initiation Complex (Step-by-Step)

• TFIID (containing TBP + TAFs) binds the TATA box, bending DNA ~80°.

• TFIIA stabilises the TBP-TATA interaction and blocks repressors.

• TFIIB bridges TBP and RNA Pol II; helps determine the TSS.

• RNA Pol II + TFIIF complex join the assembling complex.

• TFIIE joins and recruits TFIIH.

• TFIIH: contains helicase (XPB, XPD) that unwinds DNA (requires ATP) and a kinase (CDK7) that phosphorylates the C-Terminal Domain (CTD) of RNA Pol II at Ser5.

• Phosphorylation of Ser5 on the CTD releases RNA Pol II from the PIC and allows it to begin elongation.

GTF	Molecular Function	Key Feature
TFIID (TBP+TAFs)	Recognises TATA box; nucleates PIC assembly	TBP bends DNA ~80°; TAFs recognise Inr, DPE
TFIIA	Stabilises TBP-DNA; anti-repressor	Not strictly essential in vitro
TFIIB	Bridges TBP and Pol II; TSS selection	Interacts with BRE element
TFIIF	Stabilises Pol II in PIC; reduces non-specific binding	Two subunits: RAP30, RAP74
TFIIE	Recruits TFIIH; modulates helicase	Has zinc-finger domain
TFIIH	DNA helicase + CDK7 kinase activity	Also functions in nucleotide excision repair

5.2 Elongation

• After promoter clearance, RNA Pol II transitions to productive elongation.

• The CTD of RNA Pol II is phosphorylated at Ser2 (by P-TEFb/CDK9) during elongation, which recruits RNA processing machinery.

• Elongation rate: ~1,000–2,000 nucleotides per minute in eukaryotes.

• DSIF (DRB Sensitivity-Inducing Factor) and NELF (Negative Elongation Factor) cause pausing at many genes; P-TEFb phosphorylates and inactivates NELF, releasing paused polymerase.

• Promoter-proximal pausing is a major regulatory checkpoint in metazoans (e.g., Drosophila hsp70 gene).

5.3 Termination

Termination of RNA Pol II transcription is not as straightforward as in prokaryotes and is mechanistically linked to 3' end processing:

• The Cleavage and Polyadenylation Specificity Factor (CPSF) recognises the polyadenylation signal (AAUAAA) in the nascent RNA.

• CPSF recruits cleavage factors (CstF, CF Im, CF IIm) to cleave the RNA 10–30 nt downstream of the poly-A signal.

• After cleavage, Poly(A) Polymerase (PAP) adds ~200 adenosine residues.

• Two models for Pol II termination: (1) Torpedo model: XRN2 exonuclease degrades the downstream RNA, 'torpeoing' the polymerase; (2) Allosteric model: cleavage causes conformational change in Pol II.

5.4 Post-Transcriptional Modifications of mRNA

The primary RNA transcript in eukaryotes (pre-mRNA or hnRNA) undergoes extensive processing in the nucleus before export:

A. 5' Capping

• Occurs co-transcriptionally as soon as the transcript is ~20-30 nt long.

• A 7-methylguanosine (m7G) cap is added to the 5' end via an unusual 5'–5' triphosphate linkage.

• Reactions: (1) RNA 5'-triphosphatase removes the γ-phosphate; (2) Guanylyltransferase adds GMP via 5'–5' bond; (3) Guanine-N7-methyltransferase methylates the guanosine.

• Functions: Protects mRNA from 5' exonucleases; required for ribosome binding (43S complex); involved in mRNA export; enhances splicing of the first intron.

B. 3' Polyadenylation

• Most eukaryotic mRNAs acquire a poly(A) tail of ~200 adenosine residues.

• Poly-A signal: AAUAAA (6-mer) located 10-30 nt upstream of the cleavage site.

• The pre-mRNA is cleaved and a poly(A) tail is added by poly(A) polymerase (PAP).

• Functions: Protects mRNA from 3' degradation; assists nuclear export; stimulates translation initiation via PABP (Poly-A Binding Protein) interactions with eIF4G.

• Replication-dependent histone mRNAs are exceptions — they end in a stem-loop, not a poly-A tail.

C. RNA Splicing (Pre-mRNA Splicing)

• Most eukaryotic genes are interrupted by non-coding sequences called introns (intervening sequences). Coding sequences are called exons (expressed sequences).

• Introns are removed and exons are joined by a large ribonucleoprotein complex called the spliceosome.

• Conserved sequences at splice sites: 5' splice site (GU...AG) — GT-AG rule; branch point (internal A residue, ~20-50 nt upstream of 3' splice site); polypyrimidine tract (before the 3' AG).

• Splicing mechanism involves two transesterification reactions: (1) 2'-OH of branch point A attacks the 5' splice site → lariat intermediate + free 5' exon; (2) 3'-OH of free 5' exon attacks the 3' splice site → joined exons + lariat intron.

• The lariat intron is debranched and degraded.

• Alternative splicing produces different mRNA isoforms from a single gene — a major mechanism of proteome diversity. Approximately 95% of human multi-exon genes are alternatively spliced.

Modification	Details
5' Capping	m7G cap; 5'–5' triphosphate bond; protects from 5' exonucleases; aids ribosome binding
3' Polyadenylation	~200 A residues; poly-A signal AAUAAA; protects from 3' degradation; aids export
Splicing	Removal of introns by spliceosome; GT-AG rule; two transesterifications; lariat intermediate
Internal Methylation	N6-methyladenosine (m6A); affects mRNA stability, translation, splicing; 'epitranscriptomics'
mRNA Export	Cap-binding complex + NXF1:NXT1 export receptor; through nuclear pore complexes

CLINICAL CONNECTION: Mutations in splice site sequences or spliceosome components cause diseases such as spinal muscular atrophy (SMN2 splicing defect), beta-thalassemia (splice site mutations in HBB gene), and various cancers.

QUADRANT II

e-Content | Supplementary Material, Key Notes & Comparisons

Key Definitions Glossary

Term	Definition	Context
Transcription	Synthesis of RNA from a DNA template by RNA polymerase	Universal; prokaryotes & eukaryotes
Promoter	DNA sequence where RNA polymerase binds to initiate transcription	Upstream of TSS
Sigma factor (σ)	Prokaryotic subunit that confers promoter specificity to RNA polymerase	Prokaryotes only
Pribnow box	Consensus -10 element (TATAAT) in prokaryotic promoters	E. coli & related bacteria
TATA box	Eukaryotic core promoter element ~-25 bp; TATAAA; bound by TBP	RNA Pol II genes
TBP	TATA-Binding Protein; subunit of TFIID; bends DNA at TATA box	Eukaryotes
CTD	C-Terminal Domain of RNA Pol II; heptapeptide repeats; phosphorylated during transcription	Eukaryotic Pol II
Rho factor	Hexameric RNA helicase; required for Rho-dependent termination in bacteria	Prokaryotes
Spliceosome	Large snRNA+protein complex that removes introns from pre-mRNA	Eukaryotes
Pre-mRNA	Unprocessed nuclear RNA transcript; contains introns; undergoes splicing, capping, poly-A addition	Eukaryotes
m7G cap	7-methylguanosine cap added co-transcriptionally to 5' end of mRNA	Eukaryotic mRNAs
Poly-A tail	~200 adenosine residues added to 3' end of mRNA; protects from degradation	Most eukaryotic mRNAs
Intron	Non-coding intervening sequence removed by splicing; GT-AG rule at splice sites	Eukaryotic genes
Exon	Expressed coding sequence retained in mature mRNA after splicing	Eukaryotic genes
Alternative splicing	Different mRNA isoforms from one gene by including/skipping different exons	Eukaryotes; ~95% of genes
snRNA	Small nuclear RNA; component of spliceosome (U1, U2, U4, U5, U6)	Eukaryotes
Enhancer	Cis-acting DNA element that increases transcription; position and orientation independent	Eukaryotes
PIC	Pre-Initiation Complex; GTFs + RNA Pol II assembled at core promoter	Eukaryotes
Transcription bubble	Locally unwound region of DNA (~17 bp) where RNA synthesis occurs	Prokaryotes & eukaryotes
Holoenzyme	Core RNA Pol + sigma factor; complete functional RNAP in bacteria	Prokaryotes

Master Comparison: Prokaryotic vs Eukaryotic Transcription

Parameter	Prokaryotes	Eukaryotes
Site	Cytoplasm (coupled with translation)	Nucleus (uncoupled from translation)
RNA Polymerase	Single RNAP (α₂ββ'ωσ)	Pol I, II, III (+ IV, V in plants)
Promoter (-10)	Pribnow box: TATAAT	TATA box: TATAAA
Promoter (-35)	TTGACA	BRE, Inr, DPE
Promoter recognition	Direct by σ factor	Indirect via GTFs (TFIID etc.)
Initiation factors	Sigma factor (σ)	General Transcription Factors (TFIIA–H)
Primer requirement	Not required	Not required
5' modification	5'-triphosphate (pppN)	m7G cap (5'–5' bond)
3' modification	None	Poly-A tail (~200 A)
Splicing	Absent (no introns, mostly)	Extensive; by spliceosome
Termination	Rho-independent or Rho-dependent	Torpedo/allosteric; linked to poly-A
Inhibitors	Rifampicin (blocks initiation)	α-amanitin (Pol II); actinomycin D
mRNA stability	Short (minutes)	Longer (hours to days)
Operon organisation	Common (polycistronic mRNA)	Rare; mostly monocistronic
Regulation	Predominantly at initiation	Chromatin remodelling + enhancers + GTFs

Quick Summary Notes

The following summary is designed for rapid revision before examinations:

PROKARYOTIC TRANSCRIPTION IN BRIEF: 1. RNAP = α₂ββ'ω (core) + σ (sigma) = holoenzyme 2. σ70 recognises -10 (TATAAT) and -35 (TTGACA) 3. Open complex formation → RNA synthesis starts at +1 4. σ dissociates after ~9-10 nt; elongation by core enzyme 5. Termination: intrinsic (GC hairpin + poly-U) OR Rho-dependent (Rho helicase + rut site) 6. No RNA processing; translation begins before transcription ends

EUKARYOTIC TRANSCRIPTION IN BRIEF: 1. Three RNAPs: Pol I (rRNA), Pol II (mRNA), Pol III (tRNA) 2. Pol II needs GTFs: TFIID binds TATA box (via TBP) → PIC assembly 3. TFIIH phosphorylates CTD Ser5 → initiation; Ser2 → elongation 4. Post-transcriptional: 5' m7G cap + 3' poly-A tail + splicing (spliceosome; GT-AG rule) 5. Mature mRNA exported from nucleus to cytoplasm for translation

Flowchart: Prokaryotic Transcription

1. Holoenzyme (α₂ββ'ωσ) binds promoter → Closed complex

2. DNA unwinding → Open complex (transcription bubble ~17 bp)

3. NTP addition → Dinucleotide formed; abortive initiations

4. Sigma dissociates → Promoter clearance → Elongation begins

5. RNA synthesised 5'→3' at ~40-50 nt/sec; topoisomerases relieve strain

6. Terminator reached → Rho-independent OR Rho-dependent termination

7. Transcript released → mRNA ready for translation (no processing)

Flowchart: Eukaryotic mRNA Biogenesis

1. TFIID binds TATA box (TBP bends DNA) → PIC assembly (TFIIA → B → F+PolII → E → H)

2. TFIIH helicase unwinds DNA; CDK7 phosphorylates CTD Ser5 → Pol II released

3. 5' m7G capping occurs co-transcriptionally (~25-30 nt)

4. Elongation: CTD Ser2 phosphorylated by P-TEFb; pausing released by CDK9

5. Pre-mRNA spliced by spliceosome (two transesterifications; lariat intermediate)

6. CPSF recognises AAUAAA → cleavage → PAP adds poly-A tail (~200 A)

7. Mature mRNA (capped + spliced + poly-A) exported via nuclear pore → Translation

QUADRANT III

Self-Assessment | MCQs, Short & Long Questions, Assertion-Reason

Section A: Multiple Choice Questions

Choose the most appropriate answer. Answers and hints are provided.

Q1. Which subunit of prokaryotic RNA polymerase is responsible for promoter recognition?

(a) Alpha (α)

(b) Beta (β)

(d) Omega (ω)

Answer: (c) Sigma (σ)

Hint: The sigma factor confers promoter specificity; it dissociates after initiation.

Q2. The Pribnow box in E. coli is located at which position relative to the transcription start site?

(a) +10

(b) -10

(d) -25

Answer: (b) -10

Hint: The Pribnow box (TATAAT) is the -10 element; -35 element is TTGACA.

Q3. Which RNA polymerase is responsible for synthesising mRNA in eukaryotes?

(a) RNA Polymerase I

(b) RNA Polymerase II

(d) RNA Polymerase IV

Answer: (b) RNA Polymerase II

Hint: Pol I makes rRNA, Pol II makes mRNA, Pol III makes tRNA and 5S rRNA.

Q4. The TATA-binding protein (TBP) is a component of which general transcription factor?

(a) TFIIB

(b) TFIIF

(d) TFIIH

Answer: (c) TFIID

Hint: TFIID = TBP + TAFs (TBP-Associated Factors); it nucleates PIC assembly.

Q5. Which reaction does TFIIH catalyse during eukaryotic transcription initiation?

(a) Phosphorylation of sigma factor

(b) Cleavage of RNA at the 3' end

(d) Poly-A tail addition

Answer: (c) ATP-dependent DNA unwinding and CTD phosphorylation

Hint: TFIIH has XPB helicase + CDK7 kinase that phosphorylates CTD Ser5.

Q6. In Rho-independent termination, which structural feature of the RNA causes the polymerase to stall?

(a) A poly-A tail

(b) A GC-rich stem-loop followed by poly-U

(d) An m7G cap

Answer: (b) A GC-rich stem-loop followed by poly-U

Hint: The hairpin stalls RNAP; weak rU-dA base pairs destabilise the hybrid.

Q7. The 7-methylguanosine cap on eukaryotic mRNA is linked to the 5' end through which type of bond?

(a) 3'–5' phosphodiester bond

(b) 2'–5' phosphodiester bond

(d) N-glycosidic bond

Answer: (c) 5'–5' triphosphate bond

Hint: The m7G cap is added via an unusual 5'-5' triphosphate linkage, not the standard 3'-5'.

Q8. What is the consensus sequence of the polyadenylation signal in eukaryotic pre-mRNA?

(a) AAUAAA

(b) TATAAT

(d) GGGCCC

Answer: (a) AAUAAA

Hint: AAUAAA (hexamer) is recognised by CPSF; cleavage and poly-A addition occur 10-30 nt downstream.

Q9. During pre-mRNA splicing, the lariat intermediate is formed by:

(a) Cleavage at the 5' splice site by CPSF

(b) Attack of 2'-OH of the branch point A on the 5' splice site

(d) Binding of U1 snRNA to the 3' splice site

Answer: (b) Attack of 2'-OH of the branch point A on the 5' splice site

Hint: First transesterification: branch point A attacks the 5' splice site, creating the lariat + free 5' exon.

Q10. Which drug inhibits bacterial RNA polymerase by blocking the entry of NTPs into the active site?

(a) Alpha-amanitin

(b) Actinomycin D

(d) Cordycepin

Answer: (c) Rifampicin

Hint: Rifampicin binds the β subunit of bacterial RNAP, blocking the RNA synthesis channel after initiation.

Q11. The process by which different mRNA molecules are produced from a single gene is called:

(a) RNA editing

(b) Trans-splicing

(d) Polyadenylation

Answer: (c) Alternative splicing

Hint: ~95% of human multi-exon genes undergo alternative splicing, generating proteome diversity.

Q12. Which snRNA in the spliceosome base-pairs with the 5' splice site of the pre-mRNA?

(a) U2 snRNA

(b) U1 snRNA

(d) U6 snRNA

Answer: (b) U1 snRNA

Hint: U1 snRNA recognises the 5' splice site (GU); U2 snRNA recognises the branch point.

Section B: Short Answer Questions

Answer each question in 2–4 sentences (approximately 50–100 words).

SAQ 1. What is the difference between the template strand and the coding strand of DNA? Why is this distinction important in transcription?