Unit 5: RNA and Protein Synthesis and Processing

I. Orientation: The Flow of Genetic Information

Gene expression converts the DNA sequence into functional protein via RNA intermediates, first formalised as the central dogma (Crick, 1958): DNA → RNA → protein. Transcription copies one DNA strand into RNA; translation decodes RNA into polypeptide. The following sections keep returning to these defining features.

• Template and coding strands: RNA polymerase reads the template (antisense, 3'→5'); the RNA produced matches the coding (sense) strand, with U replacing T.
• Directionality: RNA is synthesised 5'→3'; nucleotides add to the free 3'-OH. No primer is required, unlike DNA replication.
• Substrate and energy: ribonucleoside triphosphates (ATP, GTP, CTP, UTP); pyrophosphate release drives chain growth.
• RNA classes: mRNA (carries codons), tRNA (adaptor, carries amino acid), rRNA (ribosome catalytic/structural core), plus regulatory small RNAs.
• Codon convention: triplet codons, 61 sense + 3 stop (UAA, UAG, UGA); start codon AUG (Met/fMet).

II. Transcription in Prokaryotes

A. A single polymerase copying operons

Bacterial transcription uses one core RNA polymerase producing polycistronic mRNAs directly coupled to translation.

• Core enzyme: subunit composition α₂ββ'ω; adds sigma (σ) factor to form the holoenzyme that recognises promoters.
• Promoter elements: consensus –10 box (Pribnow, TATAAT) and –35 box (TTGACA), spaced ~17 bp, upstream of the +1 start site.
• Initiation: σ binds the promoter; the closed complex opens (~14 bp melted) to an open complex; abortive synthesis precedes σ release (promoter clearance).
• Elongation: core enzyme moves at ~40–50 nt/s, maintaining a ~8 bp RNA–DNA hybrid within a transcription bubble.
• Termination: two modes contrasted.
  1. Rho-independent (intrinsic): a GC-rich hairpin followed by a poly-U stretch destabilises the hybrid, ejecting RNA.
  2. Rho-dependent: the Rho helicase loads on a C-rich rut site, translocates along RNA, and unwinds the hybrid at the polymerase.

III. Transcription in Eukaryotes

A. Three polymerases and assembled machinery

Eukaryotic transcription occurs in the nucleus using three specialised polymerases and general transcription factors (GTFs).

• Polymerase division of labour: Pol I → rRNA (28S, 18S, 5.8S); Pol II → mRNA and most snRNA; Pol III → tRNA, 5S rRNA.
• Core promoter (Pol II): TATA box (~–25, consensus TATAAA), Inr, and downstream elements bound via TBP.
• Pre-initiation complex (PIC): GTFs assemble in order — TFIID → TFIIA → TFIIB → TFIIF/Pol II → TFIIE → TFIIH.
• TFIIH functions: helicase activity opens DNA; its CDK7 kinase phosphorylates the Pol II C-terminal domain (CTD) Ser5, triggering promoter escape.
• Enhancers and mediator: distal enhancers bound by activators loop to the promoter; the Mediator complex bridges them to Pol II.
• Contrast with prokaryotes: transcription and translation are spatially separated; pre-mRNA requires processing before export.

IV. Antibiotic Inhibitors of Transcription

A. Blocking RNA polymerase selectively

These drugs exploit structural differences between bacterial, eukaryotic and organellar polymerases.

• Rifampicin: binds the bacterial β-subunit, blocking extension beyond ~2–3 nt; used against Mycobacterium tuberculosis.
• Actinomycin D: intercalates into GC-rich DNA, blocking elongation by any polymerase; anticancer agent, not selective.
• α-Amanitin: from Amanita phalloides; binds the bridge helix of eukaryotic Pol II (high affinity) and Pol III (low), stalling translocation.
• Streptolydigin: inhibits bacterial polymerase by blocking the catalytic nucleotide-addition step.

V. Post-Transcriptional Modifications

A. Maturing eukaryotic pre-mRNA

Nuclear pre-mRNA is capped, spliced and polyadenylated co-transcriptionally to become an exportable, stable, translatable mRNA.

B. Capping

A modified guanosine is added to the 5' end to protect and mark the transcript.

• Structure: 7-methylguanosine joined by an unusual 5'–5' triphosphate linkage (m⁷GpppN).
• Enzymatic steps: RNA triphosphatase removes γ-phosphate → guanylyltransferase adds GMP → methyltransferase (SAM donor) methylates N7.
• Timing: occurs when the transcript reaches ~25–30 nt, on the phosphorylated CTD.
• Roles: protects against 5'→3' exonucleases; recruits eIF4E for translation initiation; aids splicing and export.

C. RNA splicing

Introns are excised and exons joined by the spliceosome via two transesterification reactions.

• Consensus sequences: 5' splice site GU, 3' splice site AG (the "GT–AG rule" on DNA), branch-point A, and polypyrimidine tract.
• Spliceosome: snRNPs U1, U2, U4, U5, U6 plus proteins; U1 binds 5' site, U2 binds branch point.
• Mechanism:
  1. Branch-point A 2'-OH attacks the 5' splice site, forming a lariat.
  2. Freed exon 3'-OH attacks the 3' splice site, ligating exons and releasing the lariat intron.
• Alternative splicing: one gene yields multiple mRNAs (exon skipping, alternative sites), expanding proteome diversity.
• Self-splicing: Group I/II introns act as ribozymes without a spliceosome.

D. Polyadenylation

A poly(A) tail is added to the 3' end after endonucleolytic cleavage.

• Signal: the hexamer AAUAAA ~10–30 nt upstream of the cleavage site, plus a downstream GU-rich element.
• Machinery: CPSF recognises AAUAAA, CstF binds the GU element; cleavage occurs, then poly(A) polymerase adds ~200 A residues (no template).
• Roles: stability, nuclear export, and translation efficiency via PABP.

VI. Protein Synthesis in Prokaryotes

A. Translation on the 70S ribosome

Bacterial translation reads mRNA in three phases on a 70S ribosome (50S + 30S subunits).

• Initiation: the Shine–Dalgarno sequence (AGGAGG) pairs with 16S rRNA to position the start AUG; initiator fMet-tRNAᶠᴹᵉᵗ binds; factors IF1, IF2, IF3 assist.
• Elongation cycle:
  1. EF-Tu·GTP delivers aminoacyl-tRNA to the A site.
  2. Peptidyl transferase (23S rRNA ribozyme) forms the peptide bond.
  3. EF-G·GTP drives translocation (A→P→E sites).
• Termination: release factors RF1 (UAA/UAG), RF2 (UAA/UGA), RF3 hydrolyse the peptidyl-tRNA bond; RRF recycles the ribosome.
• Coupling: ribosomes translate mRNA while it is still being transcribed.

VII. Protein Synthesis in Eukaryotes

A. Translation on the 80S ribosome

Eukaryotic translation uses an 80S ribosome (60S + 40S) and cap-dependent scanning.

• Initiation: eIF4F (eIF4E cap-binding, eIF4G scaffold, eIF4A helicase) binds the cap; the 43S complex (40S + Met-tRNAᵢ + eIF2·GTP) scans to the first AUG in Kozak context (gccAccAUGG).
• Initiator tRNA: carries unformylated Met (not fMet).
• Elongation factors: eEF1A (aa-tRNA delivery) and eEF2 (translocation) mirror bacterial EF-Tu and EF-G.
• Termination: single factor eRF1 recognises all three stop codons; eRF3 provides GTP hydrolysis.
• Regulation: phosphorylation of eIF2α halts initiation under stress.

VIII. Inhibitors of Translation

A. Antibiotics and toxins targeting the ribosome

Selective toxicity arises from 70S vs 80S ribosome differences.

• Prokaryote-selective (antibiotics):
  • Streptomycin: binds 30S, causes misreading and blocks initiation.
  • Tetracycline: blocks aminoacyl-tRNA entry to the A site (30S).
  • Chloramphenicol: inhibits 50S peptidyl transferase.
  • Erythromycin: binds 50S, blocks translocation through the exit tunnel.
• Eukaryote-selective / both:
  • Cycloheximide: blocks 60S translocation (eukaryotic).
  • Puromycin: aminoacyl-tRNA analogue causing premature chain release in both.
  • Diphtheria toxin: ADP-ribosylates and inactivates eEF2.
  • Ricin: depurinates 28S rRNA, inactivating the 60S subunit.

IX. Post-Translational Modifications

A. Tailoring the finished polypeptide

After synthesis, proteins are chemically altered, cut, or spliced to reach their functional, localised, mature form.

B. Chemical modifications

Covalent groups added to specific residues tune activity, localisation and interactions.

• Phosphorylation: kinases add phosphate to Ser/Thr/Tyr –OH; reversible on/off switch (e.g. signalling cascades); removed by phosphatases.
• Glycosylation: N-linked to Asn (Asn-X-Ser/Thr) or O-linked to Ser/Thr; occurs in ER/Golgi; aids folding and cell recognition.
• Ubiquitination: 76-residue ubiquitin attached to Lys via E1–E2–E3 cascade; polyubiquitin (Lys48) targets the 26S proteasome.
• Acetylation and methylation: on histone Lys/Arg, altering chromatin state and gene expression.
• Lipidation: myristoylation, palmitoylation, prenylation anchor proteins to membranes.
• Disulphide bonds: Cys–Cys oxidation stabilises secreted proteins in the ER.

C. Proteolytic cleavage

Irreversible peptide-bond hydrolysis converts inactive precursors to active products.

• Signal peptide removal: signal peptidase cleaves the N-terminal ER-targeting sequence.
• Zymogen activation: inactive precursors activated by cleavage — e.g. trypsinogen → trypsin; proinsulin → insulin (removal of C-peptide).
• Polyprotein processing: viral polyproteins (e.g. HIV Gag-Pol) cut by proteases into functional units.

D. Protein splicing

Internal segments are self-excised at the protein level, analogous to RNA splicing.

• Inteins and exteins: the internal intein removes itself, ligating the flanking exteins into the mature protein.
• Mechanism: an autocatalytic four-step reaction via N–S/O acyl shifts forms a branched intermediate, then a peptide bond joins the exteins.
• Feature: requires no external enzyme or energy input; the intein often carries a homing endonuclease domain.