Translation & Post-translational Modification

Introduction to Translation

Translation is the process by which the sequence of codons in an mRNA is decoded to produce a specific polypeptide. It is the final step in the central dogma (DNA → RNA → Protein) and is carried out by the ribosome — one of the most ancient and conserved molecular machines in biology. After synthesis, proteins undergo post-translational modifications (PTMs) that dramatically expand functional diversity and regulate protein activity, localisation, and stability.

Ribosome Structure

Ribosomes are large ribonucleoprotein complexes. Eukaryotic ribosomes are 80S, consisting of:

• Small subunit (40S): 18S rRNA + ~33 proteins; decodes the mRNA; contains the decoding centre
• Large subunit (60S): 28S + 5.8S + 5S rRNA + ~49 proteins; catalyses peptide bond formation; contains the peptidyl transferase centre (PTC) — a ribozyme (the rRNA, not the protein, is catalytic; confirmed by crystal structures)

The ribosome has three tRNA-binding sites:

• A site (aminoacyl site): incoming aminoacyl-tRNA
• P site (peptidyl site): tRNA carrying the growing peptide chain
• E site (exit site): departing deacylated tRNA

The mRNA threads through a channel between the two subunits. The anti-codon of the aminoacyl-tRNA in the A site base-pairs with the mRNA codon; the peptidyl transferase centre catalyses peptide bond formation.

The Genetic Code

The genetic code assigns each of the 64 possible trinucleotide codons to an amino acid or a stop signal:

• Three stop codons: UAA, UAG, UGA (recognised by release factors, not tRNAs)
• One start codon: AUG (methionine) — also signals the start of a reading frame
• The code is degenerate (redundant): most amino acids are encoded by 2–6 codons (only Met and Trp have single codons). Synonymous codons typically differ only at the third position (wobble position) — the anticodon first base (5') can base-pair non-canonically
• The code is nearly universal: all organisms use essentially the same code (minor exceptions in mitochondria and some organisms)
• Codon bias: some organisms preferentially use certain synonymous codons corresponding to the most abundant tRNAs — relevant for recombinant protein expression (codon optimisation)

Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (aaRS) charge each tRNA with its cognate amino acid in a two-step reaction:

1. Aminoacyl-AMP intermediate: Amino acid + ATP → Aminoacyl-AMP + PPi (PPi hydrolysis drives the reaction)
2. Transfer: Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP

Twenty aaRS enzymes exist (one per amino acid). They are responsible for the identity elements of tRNAs — specific nucleotides in the acceptor stem and anticodon loop that the aaRS recognises to ensure the correct amino acid is attached to the correct tRNA. aaRS also have proofreading (editing) domains that hydrolyse incorrect aminoacyl-tRNA intermediates (e.g., threonyl-tRNA synthetase prevents serine from being incorporated at Thr codons).

Translation Initiation

Eukaryotic translation initiation is the rate-limiting step and a major point of regulation:

1. 43S pre-initiation complex (PIC) assembly: initiator Met-tRNA is loaded onto the 40S subunit with eIF2-GTP to form the 43S complex
2. mRNA activation: eIF4E (cap-binding) + eIF4A (helicase, unwinds 5' UTR secondary structure) + eIF4G (scaffold) assemble as the eIF4F complex on the m7G cap; PABP binds poly-A and eIF4G, circularising the mRNA
3. 43S scanning: the 43S PIC is recruited to the 5' cap via eIF4F and scans 5'→3' until it encounters the start codon (AUG), usually in a Kozak context (RCCAUGG for strong initiation)
4. Joining: recognition of the start codon triggers eIF2-GTP hydrolysis (GTP → GDP), release of initiation factors, and joining of the 60S subunit to form the 80S ribosome ready for elongation

Regulation: eIF2α phosphorylation (by HRI, PKR, PERK, GCN2 — the integrated stress response kinases) converts eIF2 to a competitive inhibitor of eIF2B, globally suppressing translation initiation while allowing translation of stress-response mRNAs (ATF4) with inhibitory upstream open reading frames.

Translation Elongation and Termination

Elongation cycle (per amino acid added):

1. Decoding (A site): EF-Tu (eukaryotic: eEF1A)-GTP delivers aminoacyl-tRNA to the A site; correct Watson-Crick codon-anticodon pairing triggers GTP hydrolysis, releasing EF-Tu-GDP; the aminoacyl-tRNA is fully accommodated
2. Peptide bond formation: the 2'-OH of the P-site tRNA acts as catalyst; the PTC (a ribozyme) transfers the nascent peptide from the P-site tRNA to the A-site amino acid; this is extremely fast (~100 s⁻¹) and does not require GTP
3. Translocation: EF-G (eukaryotic: eEF2)-GTP powers translocation — the ribosome moves one codon 3' along the mRNA; the previous P-site (now deacylated) tRNA moves to the E site and exits; the new peptidyl-tRNA moves to the P site; the A site is vacant, ready for the next codon. GTP hydrolysis provides directionality

Termination: when a stop codon (UAA, UAG, UGA) enters the A site, a release factor (eRF1 — recognises all three stop codons; + eRF3-GTP as GTPase stimulator) enters instead of a tRNA. eRF1 induces hydrolysis of the ester bond between the peptide and the P-site tRNA, releasing the completed polypeptide. Ribosome recycling factor (ABCE1 in eukaryotes) splits the 80S ribosome into subunits for re-use.

Signal Peptide and ER Targeting

Many proteins must be delivered to the ER lumen, Golgi, plasma membrane, or outside the cell. These enter the co-translational translocation pathway:

The N-terminal signal peptide (~16–30 hydrophobic amino acids) is recognised by the signal recognition particle (SRP) as it emerges from the ribosome. SRP pauses translation and docks the ribosome-mRNA-nascent chain complex to the SRP receptor (SR) on the ER membrane. The signal peptide is threaded into the Sec61 translocon (a protein-conducting channel), translation resumes, and the growing polypeptide is translocated into the ER lumen (or, for membrane proteins, integrated laterally into the lipid bilayer). Signal peptides are cleaved by signal peptidase in the ER lumen.

N-Glycosylation

N-glycosylation is the most common glycosylation in secreted and membrane proteins. It occurs co-translationally in the ER lumen:

1. A preassembled oligosaccharide (Glc₃Man₉GlcNAc₂) is built on the lipid carrier dolichol-pyrophosphate on the cytoplasmic face of the ER, then flipped to the luminal face
2. Oligosaccharyltransferase (OST) transfers the entire glycan en bloc to the amide nitrogen of asparagine in the N-X-S/T sequon (Asn-any except Pro-Ser/Thr) co-translationally
3. Glucose trimming and early quality control: calnexin/calreticulin (lectin chaperones) retain incompletely folded proteins until correctly folded, then allow forward transport. Misfolded glycoproteins undergo ERAD (ER-associated degradation)
4. Further processing occurs in the Golgi (trimming of mannose residues; addition of complex-type sugars)

Functions of N-glycans: protein folding (hydrophilic bulk, quality control); stability (protection from proteolysis); cell recognition; targeting (mannose-6-phosphate targets enzymes to lysosomes).

Phosphorylation

Phosphorylation is the most widespread reversible PTM. Protein kinases transfer a phosphate group from ATP to the hydroxyl of serine (most common), threonine, or tyrosine (less common but crucial in signalling) residues. Protein phosphatases remove the phosphate.

The human genome encodes ~518 kinases (~1.7% of the genome — the "kinome"), reflecting the central importance of phosphorylation. Effects of phosphorylation:

• Conformational change (allosteric activation/inhibition)
• Creation of phosphopeptide binding sites for SH2 (recognises pTyr), SH3, 14-3-3 (pSer/pThr) domain-containing proteins
• Subcellular localisation change
• Targeting for ubiquitination and proteasomal degradation

Example: EGFR autophosphorylates on tyrosine residues upon ligand binding → recruits GRB2/SOS → RAS activation → proliferation. EGFR mutations (L858R, exon 19 deletions) cause constitutive kinase activity → lung cancer. Targeted by EGFR tyrosine kinase inhibitors (gefitinib, erlotinib, osimertinib).

Ubiquitination and the Proteasome

Ubiquitin is a small (76 aa) protein that is covalently attached to substrate lysine residues as a signal for proteasomal degradation or other fates. Three-enzyme cascade:

1. E1 (ubiquitin-activating enzyme): activates ubiquitin in an ATP-dependent reaction, forming a high-energy E1-Ub thioester
2. E2 (ubiquitin-conjugating enzyme): receives Ub from E1; works with E3
3. E3 (ubiquitin ligase): provides substrate specificity; transfers Ub to the substrate lysine. >600 E3 ligases in the human genome — the major specificity determinants

Polyubiquitin chains linked through Lys48 (K48) target substrates for the 26S proteasome — a barrel-shaped protease complex (20S catalytic + 19S regulatory caps). The 19S cap recognises polyUb chains, unfolds and deubiquitinates the substrate, and threads it into the 20S chamber for proteolysis to short peptides (~3–25 aa), which are released and recycled to free amino acids by cytoplasmic peptidases.

Bortezomib (Velcade): a proteasome inhibitor (reversibly inhibits the threonine protease activity of the 20S β5 subunit). Approved for multiple myeloma and mantle cell lymphoma. Mechanism of anti-tumour activity: myeloma cells produce massive amounts of immunoglobulin → high ER stress → depend on robust ERAD and proteasome function to clear misfolded proteins → proteasome inhibition causes proteotoxic stress and apoptosis (via accumulation of IκBα, p21, and misfolded proteins that trigger UPR → apoptosis).