Transcription & RNA Processing

Transcription is the synthesis of RNA from a DNA template by DNA-dependent RNA polymerases. In eukaryotes the primary transcript (hnRNA) is heavily processed — capped, polyadenylated and spliced — before it becomes mature, exportable mRNA. This is a favourite zone for "match the polymerase / inhibitor / RNA" MCQs in NEET PG.

Central dogma and direction

The flow of genetic information: DNA → (transcription) → RNA → (translation) → Protein, with reverse transcription (RNA → DNA) as the special exception seen in retroviruses and telomerase.

Key conventions you are repeatedly tested on:

• RNA is synthesised 5′ → 3′ (like DNA synthesis), reading the template strand 3′ → 5′.
• The template (antisense) strand is read; the coding (sense) strand has the same sequence as the mRNA except T is replaced by U.
• No primer is required (unlike DNA replication), and RNA polymerase has no proof-reading 3′→5′ exonuclease activity — hence transcription is more error-prone than replication.

High-yield: RNA polymerase does NOT need a primer and lacks proof-reading exonuclease activity. DNA polymerase needs a primer and proof-reads. This single contrast is asked very often.

Eukaryotic RNA polymerases (I, II, III)

There are three nuclear RNA polymerases in eukaryotes, distinguished by location, products and sensitivity to α-amanitin (the toxin of Amanita phalloides, the death-cap mushroom).

Polymerase	Location	Products	α-amanitin sensitivity
RNA Pol I	Nucleolus	45S pre-rRNA → 28S, 18S, 5.8S rRNA	Resistant (insensitive)
RNA Pol II	Nucleoplasm	mRNA (hnRNA), most snRNA, miRNA	Very sensitive (low dose inhibits)
RNA Pol III	Nucleoplasm	tRNA, 5S rRNA, U6 snRNA	Sensitive only to high dose

High-yield: RNA Pol II is the most α-amanitin-sensitive and makes mRNA. Pol I (rRNA, nucleolus) is resistant. Death-cap poisoning kills mainly through hepatic Pol II inhibition.

Mnemonic — "I make ribosomes, II make messengers, III make transfers": Pol I → rRNA, Pol II → mRNA, Pol III → tRNA. Also: the numbers go I-II-III as you move from nucleolus → nucleoplasm.

Prokaryotes have a single RNA polymerase (core enzyme α₂ββ′ω + sigma (σ) factor = holoenzyme). Sigma recognises the promoter; once initiation occurs, sigma dissociates and the core enzyme elongates. Rho (ρ) factor mediates one form of termination.

Promoters and cis-acting elements

Promoters are DNA sequences upstream of the start site that position the polymerase.

Prokaryotic promoter:

• −10 Pribnow box (consensus TATAAT)
• −35 box (consensus TTGACA)

Eukaryotic Pol II promoter:

• TATA box (Hogness box) ~ −25 to −30, bound by TBP (TATA-binding protein), a subunit of TFIID.
• CAAT box and GC box further upstream.
• Enhancers — act at a distance, in either orientation, bound by activators; silencers repress.

General transcription factors for Pol II assemble in order: TFIID → TFIIA → TFIIB → TFIIF (+Pol II) → TFIIE → TFIIH. TFIIH has helicase and kinase activity that phosphorylates the CTD (carboxy-terminal domain) of Pol II to trigger promoter clearance.

High-yield: TBP (within TFIID) binds the TATA box; TFIIH phosphorylates the Pol II CTD to start elongation. CTD phosphorylation also coordinates capping, splicing and polyadenylation.

Stepwise transcription cycle: Initiation → Promoter clearance → Elongation → Termination, then (in eukaryotes) co-transcriptional processing.

The three classic RNA-processing events (Pol II / mRNA)

Processing of the primary transcript (heterogeneous nuclear RNA, hnRNA) happens co-transcriptionally in the nucleus.

1. 5′ Capping

• A 7-methylguanosine (m⁷G) cap is added to the 5′ end via an unusual 5′→5′ triphosphate linkage.
• Enzymes: RNA triphosphatase → guanylyltransferase → guanine-7-methyltransferase.
• Functions: protects mRNA from 5′ exonucleases, aids nuclear export, and is recognised by eIF4E for ribosomal 40S binding during translation initiation.

2. 3′ Polyadenylation

• The signal AAUAAA (≈10–30 nt upstream of the cleavage site) is recognised by CPSF; the transcript is cleaved and a poly-A tail (~100–250 adenines) added by poly-A polymerase (template-independent).
• Functions: stability and export; tail length shortens with age of the mRNA.

3. Splicing — removal of introns

• Introns (intervening, non-coding) are removed; exons (expressed) are ligated.
• Splice sites follow the GU–AG rule (GT–AG in DNA): introns begin with GU at the 5′ donor and end with AG at the 3′ acceptor.
• A conserved branch-point adenine (A) within the intron performs the first nucleophilic attack.

Spliceosome: a complex of snRNPs (small nuclear ribonucleoproteins) — U1, U2, U4, U5, U6 (often pronounced "snurps").

• U1 binds the 5′ donor splice site.
• U2 binds the branch point.
• U6 participates in catalysis (the spliceosome's RNA is catalytic).

Two trans-esterification reactions:

1. Branch-point A's 2′-OH attacks the 5′ splice site → forms a lariat intermediate.
2. Freed 5′ exon attacks the 3′ splice site → exons joined, lariat (intron) released.

High-yield: GU-AG rule + branch-point adenine + lariat formation = classic spliceosomal splicing. Spliceosomal snRNAs (U1–U6, except U3) are transcribed by Pol II; U6 by Pol III.

Processing step	Signal / machinery	Key fact
5′ cap	m⁷G, 5′→5′ link	Bound by eIF4E; protects from exonuclease
Poly-A tail	AAUAAA + CPSF + poly-A polymerase	Template-independent, ~200 A residues
Splicing	snRNPs U1,U2,U4,U5,U6; GU-AG	Lariat via 2 trans-esterifications

Ribozymes — catalytic RNA

RNA molecules with enzymatic activity. Thomas Cech and Sidney Altman won the 1989 Nobel Prize for discovering catalytic RNA.

• Self-splicing introns — Group I (e.g. Tetrahymena rRNA precursor; uses an external guanosine cofactor) and Group II (use an internal branch-point A, mechanism resembling spliceosomal splicing — supports the idea that the spliceosome is RNA-based).
• RNase P — processes the 5′ end of tRNA; its RNA subunit is the catalytic component.
• Peptidyl transferase in the large ribosomal subunit (28S/23S rRNA) — the ribosome is itself a ribozyme.

High-yield: The ribosome's peptidyl transferase activity resides in the rRNA, not protein — the ribosome is a ribozyme. Group I introns need an external G; Group II resemble spliceosomes.

Types of RNA — quick reference

RNA	Made by	Function / note
mRNA	Pol II	Carries code; ~2–5% of cell RNA; shortest-lived
rRNA	Pol I (5S by Pol III)	Most abundant cellular RNA; structural + catalytic
tRNA	Pol III	Adaptor; ~75 nt; has anticodon + CCA 3′ end; most modified bases
snRNA	Pol II (U6 by Pol III)	Splicing (U1,2,4,5,6)
snoRNA	—	Guides rRNA chemical modification in nucleolus
miRNA	Pol II	~22 nt; gene silencing, blocks translation
siRNA	exogenous/dsRNA	RNA interference, mRNA degradation
hnRNA	Pol II	Unprocessed nuclear precursor of mRNA

High-yield: rRNA is the most abundant RNA in the cell; tRNA contains the most modified/unusual bases (e.g. pseudouridine, dihydrouridine, inosine). mRNA is least abundant and least stable.

miRNA, siRNA and gene silencing

RNA interference (RNAi) — sequence-specific gene silencing by small RNAs. Fire and Mello won the 2006 Nobel Prize for RNAi.

miRNA pathway flow: pri-miRNA (Pol II) → Drosha (nuclear microprocessor) → pre-miRNA → exported by Exportin-5 → Dicer (cytoplasm) → mature miRNA → loaded onto RISC (Argonaute) → binds 3′-UTR of target mRNA → translational repression / degradation.

• miRNA usually binds with imperfect complementarity → represses translation.
• siRNA binds with perfect complementarity → cleaves/degrades target mRNA.
• Both use Dicer and the RISC complex (Argonaute protein).

High-yield: Drosha acts in the nucleus, Dicer in the cytoplasm; Exportin-5 shuttles pre-miRNA out. RISC contains Argonaute. siRNA = perfect match → degrade; miRNA = imperfect → repress.

Reverse transcription and special enzymes

• Reverse transcriptase (RNA-dependent DNA polymerase) — in retroviruses (HIV); target of NRTIs/NNRTIs. Has RNase H activity.
• Telomerase — a reverse transcriptase carrying its own RNA template; adds TTAGGG repeats to chromosome ends; reactivation is a hallmark of cancer cells; absent/low in most somatic cells (contributes to ageing/Hayflick limit).

Inhibitors of transcription — heavily tested pharmacology overlap

Drug / toxin	Mechanism	Use / relevance
Rifampicin	Inhibits prokaryotic RNA polymerase (β subunit), blocks initiation	Anti-TB; selective for bacteria
α-Amanitin	Inhibits eukaryotic RNA Pol II (and III at high dose)	Death-cap mushroom hepatotoxicity
Actinomycin D (dactinomycin)	Intercalates GC, blocks template; high dose stops all RNA synthesis, low dose blocks rRNA	Antineoplastic (Wilms tumour, choriocarcinoma)
Doxorubicin / daunorubicin	Intercalation + topoisomerase II poison	Antineoplastic
Fluoroquinolones	Inhibit DNA gyrase/topo IV (DNA, not RNA Pol)	(contrast/distractor)
Amatoxin antidote	Silibinin ± penicillin G	Supportive

High-yield: Rifampicin = bacterial RNA polymerase; α-amanitin = eukaryotic Pol II; Actinomycin D intercalates DNA and at low dose preferentially blocks rRNA (Pol I). This trio is a perennial single-best-answer set.

Prokaryotic vs eukaryotic transcription — comparison

Feature	Prokaryotes	Eukaryotes
RNA polymerase	One (core + σ)	Three (Pol I, II, III)
Location	Cytoplasm (coupled to translation)	Nucleus (separate from translation)
Coupling	Transcription & translation simultaneous	Uncoupled (nuclear membrane)
Promoter	−10 Pribnow, −35 box	TATA/Hogness, CAAT, GC boxes
Capping/polyA/splicing	Absent	Present
mRNA	Polycistronic	Mostly monocistronic
Initiation factor	σ factor	TFIID (TBP) + GTFs
Termination	Rho-dependent / intrinsic hairpin	Cleavage + polyadenylation linked

Alternative splicing and clinical correlates

Alternative splicing lets one gene encode multiple protein isoforms (e.g. calcitonin vs CGRP from the same gene; membrane vs secreted antibody). It explains how ~20,000 human genes produce a far larger proteome.

Disease links worth remembering:

• β-Thalassaemia — many mutations affect splice sites of the β-globin gene → aberrant splicing.
• Systemic lupus erythematosus (SLE) — anti-Sm antibodies target snRNP core proteins (highly specific for SLE); anti-U1-RNP is seen in mixed connective tissue disease.
• Spinal muscular atrophy — SMN gene, involved in snRNP assembly.
• Xeroderma pigmentosum / trichothiodystrophy — defects in TFIIH subunits (XPB, XPD) link transcription with nucleotide-excision repair.

High-yield: Anti-Sm antibodies (against spliceosomal snRNP proteins) are highly specific for SLE — a frequent cross-link between biochemistry and immunology/medicine MCQs.

Recently asked / exam angle

• Match RNA polymerase → product → α-amanitin sensitivity (Pol II / mRNA / most sensitive).
• Which RNA polymerase synthesises tRNA / 5S rRNA → Pol III.
• Cap structure linkage type → 5′→5′ triphosphate, m⁷G.
• Splice site consensus → GU at 5′ donor, AG at 3′ acceptor; branch-point adenine; lariat formation.
• Enzyme processing the 5′ end of tRNA → RNase P (a ribozyme).
• Drug inhibiting bacterial RNA polymerase → rifampicin; eukaryotic → α-amanitin.
• Nuclear vs cytoplasmic step of miRNA: Drosha (nucleus), Dicer (cytoplasm), Exportin-5 transport.
• Most abundant RNA → rRNA; RNA with most modified bases → tRNA.
• Ribozyme / catalytic RNA examples; peptidyl transferase is rRNA.
• Anti-Sm antibody target → snRNP (SLE).
• TATA-binding protein is part of which factor → TFIID.

Rapid revision

1. RNA synthesis is 5′→3′, no primer, no proof-reading — more error-prone than replication.
2. Pol I → rRNA (nucleolus, α-amanitin resistant); Pol II → mRNA (most sensitive); Pol III → tRNA + 5S rRNA + U6.
3. Rifampicin blocks bacterial RNA polymerase; α-amanitin blocks eukaryotic Pol II.
4. Three mRNA processing events: 5′ m⁷G cap, 3′ poly-A tail (AAUAAA → CPSF), splicing.
5. The cap has a unique 5′→5′ triphosphate linkage and is read by eIF4E.
6. Splicing follows the GU–AG rule with a branch-point adenine forming a lariat via two trans-esterifications.
7. Spliceosome snRNPs = U1, U2, U4, U5, U6; U1 binds 5′ site, U2 the branch point.
8. Ribozymes = catalytic RNA: RNase P, self-splicing introns, ribosomal peptidyl transferase (Cech & Altman, 1989 Nobel).
9. miRNA: Drosha (nucleus) → Exportin-5 → Dicer (cytoplasm) → RISC; imperfect match = repress, siRNA perfect match = degrade.
10. rRNA is most abundant; tRNA has the most unusual/modified bases and a 3′ CCA end.
11. TBP/TFIID binds the TATA box; TFIIH phosphorylates the Pol II CTD and overlaps with NER (XP).
12. Anti-Sm (anti-snRNP) antibody is specific for SLE; β-thalassaemia often arises from splice-site mutations.