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'$\to$3', 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 + $\ge$200 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.