Transcription & RNA

Transcription & RNA Processing

Transcription is the synthesis of RNA from a DNA template, catalyzed by RNA Polymerase. It follows the Central Dogma: DNA → RNA → Protein.

General Mechanism

  1. Initiation: RNA polymerase binds to Promoter sequence (consensus: TATA box in eukaryotes at -25, CAAT or GC box at -75). In prokaryotes: σ (sigma) factor recognizes -10 (Pribnow box) and -35 boxes. In eukaryotes: General Transcription Factors (GTFs) + Mediator + RNA Pol II assemble at promoter.
  2. Elongation: RNA pol moves along template strand (3'→5'), synthesizes RNA in 5'→3' direction. No primer needed. Ribose nucleotides used (not deoxy). U replaces T in RNA.
  3. Termination: Prokaryotes: Rho-independent (hairpin + poly-U sequence) or Rho-dependent (Rho helicase chases RNA pol). Eukaryotes: Cleavage at poly-A signal (AAUAAA) → polyadenylation.

RNA Polymerases (Eukaryotes)

  • RNA Pol I: Nucleolus; synthesizes pre-rRNA (28S, 18S, 5.8S). Inhibited by α-Amanitin (high dose — mushroom toxin)
  • RNA Pol II: Nucleoplasm; synthesizes pre-mRNA (→mRNA), snRNA, miRNA. Inhibited by α-Amanitin (low dose — most sensitive)
  • RNA Pol III: Nucleoplasm; synthesizes tRNA, 5S rRNA, snRNA. Inhibited by α-Amanitin (very high dose)

Pre-mRNA Processing (Eukaryotes)

  • 5' Capping: 7-methylguanosine cap added co-transcriptionally. Protects from 5' exonuclease; required for ribosome binding (translation initiation); required for nuclear export.
  • 3' Polyadenylation: ~200 A residues added to 3' end after cleavage at AAUAAA signal. Protects from 3' degradation; required for nuclear export; ↑mRNA stability.
  • Splicing: Removal of introns (non-coding) and joining of exons. Site: Spliceosome (snRNPs: U1, U2, U4, U5, U6). Branch point A (within intron) attacks 5' splice site → lariat intermediate → 3' splice site attacked → intron released as lariat → exons joined. Alternative splicing: one pre-mRNA → multiple protein isoforms. Mutations in splice sites → human diseases (e.g., β-thalassemia).

Types of RNA

  • mRNA (~5%): Carries genetic code; poly-A tail; translated on ribosomes
  • rRNA (~80%): Most abundant; ribosomal scaffold + catalytic function (peptidyl transferase activity of 23S/28S rRNA = ribozyme)
  • tRNA (~15%): Aminoacid adapter; 73–93 nt; CCA-3' end; anticodon loop; L-shaped tertiary structure
  • snRNA: In spliceosome
  • miRNA: ~22 nt; base-pairs with 3'UTR of target mRNA → mRNA degradation or translation repression. Important in development and cancer.
  • siRNA: Gene silencing (RNAi); therapeutic applications
  • lncRNA: >200 nt non-coding; chromatin remodeling, X-inactivation (Xist)

Ribozymes

RNA molecules with catalytic activity. Examples: Peptidyl transferase (23S/28S rRNA), Ribonuclease P (tRNA processing), Group I and II self-splicing introns, Hepatitis Delta Virus ribozyme. Important evidence for RNA World hypothesis.

Reverse Transcription

Retroviruses (HIV): ssRNA → dsDNA via Reverse Transcriptase (RNA-dependent DNA polymerase). RT lacks proofreading → high mutation rate. NRTIs (Zidovudine/AZT) and NNRTIs target RT. Telomerase is also a reverse transcriptase.