Transcription & Translation – Comprehensive Bullet-Point Notes

Central Dogma – Information Flow

  • DNA \to RNA \to Polypeptide (protein).
  • Each level is a linear polymer whose monomer order encodes information:
    • DNA/RNA: sequence of nucleotides (A, T/U, G, C).
    • Protein: sequence of amino acids.
  • A “gene” is a DNA segment that is transcribed; its RNA product may or may not be translated.
  • Genetic information transfer is unidirectional under normal cellular conditions (reverse transcription is an exception not treated here).

Transcription – Universal Principles

  • Purpose: copy genetic information from DNA into complementary RNA.
  • Enzyme: DNA-dependent RNA polymerase (RNAP).
    • Reads the DNA template strand 3' \to 5'; synthesises RNA 5' \to 3'.
    • Uses NTPs: ATP, UTP, GTP, CTP.
    • No primer required (contrasts with DNA polymerase).
  • Occurs in three kinetic/structural phases:
    1. Initiation – promoter recognition, DNA melting, first phosphodiester bonds.
    2. Elongation – RNAP traverses gene, extends RNA, maintains a transcription bubble of \approx 14 bp.
    3. Termination – RNAP disengages, RNA released.
  • Highly regulated: cells transcribe only a subset of genes at any moment.

Bacterial (Prokaryotic) RNAP – Composition & Structure

  • Core enzyme: \alpha_2 \beta \beta' \omega (5 subunits) ≈ “crab-claw” shape (Thermus aquaticus PDB 1HQM).
  • Sigma (\sigma) factor attaches to core to form holo-enzyme and confers promoter specificity.
    • Different \sigma factors recognise distinct promoter classes (heat-shock, flagellar genes, etc.).
    • Table excerpt (Kd and cellular abundance): \sigma^{70} is the housekeeping form (≈70\% of holoenzymes).

Bacterial Promoters & \sigma-Factor Logic

  • Promoter spans roughly -70 to +30 relative to transcription start site (+1).
  • Regions:
    • UP element (very A/T-rich) binds \alpha subunit.
    • -35 and -10 consensus boxes recognised by \sigma.
  • Affinity of holo-enzyme to promoter proportional to complementarity of these elements and UP interactions.

Bacterial Transcription Cycle

  1. Initiation
    • Holo-enzyme binds promoter \Rightarrow “closed complex”.
    • Local unwinding (~14 bp, ~1 DNA turn) \Rightarrow “open complex/transcription bubble”.
  2. Elongation
    • After ~10 nt, \sigma released, replaced by NusA.
    • RNAP maintains ~8 bp RNA–DNA hybrid inside bubble.
  3. Termination
    • Rho-dependent: ρ (hexameric ATP-dependent helicase) binds rut sites (C-rich, G-poor), translocates 5'\to3', unwinds hybrid when catches RNAP.
    • Rho-independent: GC-rich palindrome \to RNA hairpin followed by U run; weak A=U pairing plus hairpin stalls RNAP, hybrid dissociates.

Transcriptional Regulation in Bacteria

  • Specificity factors (alternative \sigma) redirect RNAP.
  • Activators raise RNAP-promoter affinity (e.g., CRP–cAMP at lac operon).
  • Repressors block access (lacI on lac operator).
  • Modes (negative & positive regulation) depend on whether effector binding triggers association or dissociation of regulator.
    • Four canonical schemes: two negative, two positive (see schematic in transcript).

Example – lac Operon Logic

  • Genes: lacZ (β-galactosidase), lacY (permease), lacA (acetyl-transferase).
  • lacI repressor blocks operator; allolactose (inducer) frees DNA.
  • CRP–cAMP binds upstream to activate under low glucose.

Eukaryotic Transcription – Overview

  • Separated from translation by nuclear envelope; includes assembly, initiation, elongation, termination.
  • Three nuclear RNAPs:
    1. Pol I – pre-rRNA (18S, 5.8S, 28S).
    2. Pol II – pre-mRNA + some sn/sno/miRNAs (most regulated).
    3. Pol III – tRNA, 5S rRNA, small RNAs.
  • All require dozens of general transcription factors (GTFs).

RNA Polymerase II Initiation (TATA Promoter Paradigm)

  1. Assembly/Pre-initiation Complex (PIC)
    • TBP (part of TFIID) binds TATA box \Rightarrow sharp DNA bend.
    • TFIIA + TFIIB stabilise TBP–DNA; TFIIB provides RNAP docking site.
    • TFIIF escorts Pol II; TFIIE and TFIIH close PIC.
  2. Initiation
    • TFIIH helicase unwinds promoter using ATP.
    • TFIIH kinase phosphorylates \text{CTD}_{(Ser^5)} of Pol II (multiple heptad repeats YSPTSPS).
    • Phosphorylation \Rightarrow conformational change, promoter escape.
    • After ~60–70 nt, TFIIE then TFIIH leave; Pol II enters elongation.
  3. Elongation
    • Elongation factors (e.g., P-TEFb, SII/TFIIS, Elongin) bind CTD; enhance processivity, proof-reading.
  4. Termination
    • Mechanism less defined; involves cleavage/polyadenylation factors; Pol II CTD de-phosphorylated for recycling.

mRNA Maturation (Eukaryotes)

  • 5' Capping
    • Occurs co-transcriptionally after ~20–30 nt.
    • 7-Me-Guanosine linked 5'–5' triphosphate; first two riboses often 2'-O-methylated.
    • Functions: protect from 5' exonucleases, recruit cap-binding complex (CBC) & translation factor eIF4E.
  • Splicing
    • Removes introns, joins exons; can be co- or post-transcriptional.
    • Two mechanisms:
    1. Self-splicing Group I introns – guanosine nucleophile; intron released linear.
    2. Self-splicing Group II / Spliceosome – A-branch nucleophile forms lariat.
    • Spliceosome: snRNAs U1, U2, U4, U5, U6 + \ge200 proteins; parallels Group II chemistry.
  • Poly(A) Tail
    • Added post-transcriptionally at cleavage site 10–30 nt downstream of AAUAAA signal.
    • Length \approx 80–250 nt; bound by PABP; stabilises mRNA and aids translation initiation via circularisation.

Translation – Fundamentals

  • Converts mRNA information into polypeptide (ribosome-directed protein synthesis).
  • Major components:
    • mRNA template (with start AUG, ORF, stop codon).
    • Ribosome (rRNA + proteins): 70S (30S+50S) in bacteria; 80S (40S+60S) in eukaryotes.
    • Amino acids + tRNAs (anticodon loop + AA acceptor arm).
    • Aminoacyl-tRNA synthetases (ARS): 20 enzymes
      \text{AA} + \text{tRNA} + ATP \xrightarrow{Mg^{2+}} \text{AA–tRNA} + AMP + PP_i.
  • The genetic code:
    • Triplet codons; almost universal.
    • Start: AUG (Met). Stop: UAA (ochre), UAG (amber), UGA (opal).
    • Wobble at third codon base allows one tRNA to decode multiple codons; inosine (I) pairs with U,C,A.
    • Variants: mitochondrial code reassigns several codons; rare 21st & 22nd amino acids: selenocysteine (Sec, UGA) & pyrrolysine (Pyl, UAG) via recoding signals.

Charging & Special Initiator tRNA

  • Bacterial initiator tRNA^{fMet} first acylated with Met by MetRS, then formylated; ensures decoding only at P-site start.

Bacterial Translation Initiation Steps

  1. 30S Pre-initiation Complex
    • 30S + IF1 (blocks A-site) + IF3 (prevents premature 50S joining) bind mRNA Shine–Dalgarno (SD) sequence (16S rRNA base-pairing: 3'-!!\text{CCUCCUUA}).
  2. fMet-tRNA^{fMet} Delivery
    • IF2–GTP brings initiator tRNA to P-site, pairs with start AUG.
  3. 50S Joining & GTP Hydrolysis
    • Dissociation of IF1–3; 70S initiation complex ready for elongation.

Eukaryotic Translation Initiation

  • Much more factor-rich; mRNA circularisation enhances efficiency.
  1. 43S Pre-initiation Complex
    • 40S + eIF1, eIF1A, eIF3, eIF5 + Met-tRNA^{Met}–eIF2–GTP.
  2. mRNA Activation (eIF4F Complex)
    • eIF4E (cap-binding), eIF4A (RNA helicase, ATP-dependent), eIF4G (scaffold) bind 5' cap.
    • eIF4G bridges to PABP at poly(A) tail \Rightarrow closed-loop mRNP; associates with 43S.
  3. Scanning – 48S complex moves 5' \to 3' until it finds “Kozak” AUG (consensus GCCRCCAUGG).
  4. 60S Joining – mediated by eIF5B–GTP; eIFs released upon GTP hydrolysis \Rightarrow 80S.

Elongation Cycle (Bacteria)

  1. Decoding – EF-Tu–GTP delivers AA-tRNA to A-site. Correct codon pairing triggers ribosomal GTPase activity.
  2. Peptidyl Transfer – catalysed by 23S rRNA (peptidyl-transferase centre). A-site \alpha-NH_2 attacks P-site carbonyl \Rightarrow peptide bond.
  3. Translocation – EF-G–GTP shifts mRNA–tRNA complex one codon; de-acylated tRNA exits via E-site.

Termination & Ribosome Recycling

  • Stop codon in A-site recognised by RF1 (UAA, UAG) or RF2 (UAA, UGA).
  • RFs promote hydrolysis of peptidyl-tRNA bond releasing polypeptide.
  • RF3 + EF-G + RRF dissociate ribosome into subunits; IF3 binds 30S to prevent re-association until next round.

Comparative Snapshot – Coupling vs Compartmentalisation

  • In bacteria, transcription and translation are coupled (polysomes forming on nascent RNA; EM micrograph in E. coli showed increasing polysome size downstream of RNAP).
  • Eukaryotes physically separate processes; mRNA processing in nucleus precedes cytoplasmic translation.

Practical / Philosophical Notes

  • Regulation at transcriptional level enables rapid economy of resources; finer control at splicing and translation further diversifies proteome (alternative splicing, miRNA repression).
  • Error rates: RNAP ≈10^{-5}, ribosome ≈10^{-4} per codon; proofreading (EF-Tu timing, splicing fidelity) crucial for proteostasis.
  • Antibiotics exploit structural differences (rifampicin targets bacterial RNAP; macrolides bind 50S; aminoglycosides disrupt decoding).

Numerical & Biochemical Highlights

  • DNA unwinding in bacterial initiation: \sim14 \text{ bp}.
  • Pol II CTD: \sim 52 heptad repeats in humans.
  • Typical poly(A) tail: 80–250 \text{ nt}; bacterial mRNAs generally lack poly(A) tails of comparable stabilising length.
  • Peptidyl transfer rate in bacteria: \sim 20 \text{ aa s}^{-1} at 37^\circ\text{C}; in eukaryotes \sim 5 \text{ aa s}^{-1}.

Helpful Multimedia (Optional Review)

  • Bacterial transcription animation: https://youtu.be/tMr9XH64rtM
  • Full lecture (transcription/translation): https://youtu.be/apP5SWitnyw?t=2145
  • Eukaryotic transcription detailed: https://youtu.be/ugMJrhQSfm8
  • Translation visualised (“hippie”): https://www.youtube.com/watch?v=u9dh00iCLww

Study Tips & Connections

  • Compare promoter elements (−10/−35 vs TATA) and initiation factors (IF vs eIF) to frame prokaryote/eukaryote distinctions.
  • Practice reading codon table; memorise stop codons and wobble rules.
  • Relate spliceosomal mechanism to Group II self-splicing to appreciate evolutionary continuity.
  • Work through mechanism arrows: nucleophilic attacks in splicing & peptide bond formation to cement chemical logic.