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Translation

Core concept

  • mRNA is decoded by ribosomes to produce a polypeptide chain

Ribosome sites (three compartments)

  • A site (aminoacyl site): charged tRNA resides
  • P site (peptidyl site): growing polypeptide chain resides
  • E site (exit site): uncharged tRNA leaves

tRNA identity

tRNA is determined by:

  • T\(\Psi\)C loop
  • anticodon
  • D loop

Amino acids (basic structure)

  • central carbon with:

  • amine group

  • carboxyl group
  • H atom
  • R group

Aminoacyl-tRNA synthetase (charging tRNA)

Two-step logic :

  1. activation:

  2. amino acid carboxyl group is linked to the \(\alpha\) phosphate of ATP

  3. forms aminoacyl-AMP

  4. conjugation:

  5. breaks the aminoacyl-AMP bond just formed

  6. attaches the amino acid to the 3' end or 2' end of the A on the tRNA

Genetic code properties

Degeneracy

  • the genetic code is degenerate
  • therefore, you cannot uniquely recover the exact DNA sequence from a protein sequence

Wobble hypothesis

  • the 3' base of the codon (wobble position) can accommodate non–Watson-Crick pairing
  • enabled by tRNA flexibility

Inosine (hypoxanthine, I) note :

  • inosine (I) is derived from adenine in tRNA
  • can match with any base except guanine (example given: I–A)

Translation stages

Initiation (focus: what eIF5 does)

Ribosome binding and scanning:

  • ribosome scans from the 5' cap to find the start codon AUG
  • initiator Met-tRNA is different from elongator Met-tRNA

Steps :

  1. ribosome splits into 40S and 60S subunits
  2. eIF2 binds GTP and Met-tRNA
  3. binds to 40S to form the 43S complex
  4. eIF4 binds to the 5' cap
  5. 43S attaches to eIF4

  6. scanning:

  7. eIF1 acts as the motor and advances 3 nt at a time

  8. start codon found:

  9. hydrolysis of eIF2-bound GTP and release of P\(_i\)

  10. 60S joins
  11. eIF5 hydrolyzes and releases all factors
  12. eIF5 ensures Met-tRNA sits on the P site

Elongation

High-level cycle :

  • charged tRNA enters A site
  • peptide bond forms; chain transfers to A-site tRNA
  • tRNAs shift (translocation)
  • empty tRNA drops off

Key catalytic point:

  • 28S rRNA has peptidyl transferase activity

Elongation factors:

  • eEF1 is required for:

  • tRNA–mRNA binding

  • peptide bond formation
  • eEF2 is required for moving tRNAs backward (translocation)

Termination

Stop codons:

  • UAA, UAG, UGA

Release factors :

  1. eRF1 enters the A site
  2. polypeptide is cleaved off
  3. eRF3 hydrolyzes GTP and kicks everything off

Polypeptide directionality and organization

  • the product has:

  • 5' end as amino terminus

  • 3' end as carboxyl terminus
  • amino terminus is first translated
  • coding sequence is linear and non-overlapping

  • multiple ribosomes can translate the same mRNA simultaneously:

  • polysome

Folding and quality control

Misfolding

  • misfolding may occur due to hydrophobic interactions (skipping steps)
  • multiple misfolded proteins can aggregate

Chaperones

  • chaperones aid correct folding
  • can unfold misfolded proteins if not aggregated

GroEL–GroES chaperonin system :

  • forms a tunnel
  • polypeptide enters
  • cap closes when chain is fully inside
  • releases correctly folded protein

Heat shock proteins :

  • human chaperones that help refold heat-denatured proteins

Post-translational modifications

  • phosphorylation: adds phosphate; changes shape; on/off switch
  • glycosylation: adds sugar; helps folding
  • acetylation
  • disulfide bonds
  • ubiquitination: adds \(\ge 4\) ubiquitin groups; marks for degradation
  • lipidation: adds lipid for membrane association