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Protein Translation Mechanisms and Ribosome Function

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Translation Requirements

Requirements for translation:

  • Mature mRNA template
  • Amino acids (aa)
  • Enzyme systems
  • Energy sources (ATP, GTP)
  • Mature tRNA
  • Mature rRNA and fully assembled ribosomes (ribosomal proteins)

Genetic Code Characteristics

Characteristics of the genetic code:

  • Almost universal
  • Unambiguous (each codon specifies a single amino acid)
  • Read sequentially in the 5′ → 3′ direction
  • Nonoverlapping reading frames
  • Single reading frame per coding region
  • Specific codons for amino acids
  • Degenerate: multiple synonymous codons code for the same amino acid
  • Codons with similar sequences often encode chemically similar amino acids

Ribosomes and Structure

Ribosome subunits and features:

  • Small subunit: aligns mRNA and tRNA during decoding.
  • Large subunit: contains surface features (ridges, valleys, central protuberance, stem) and the peptide exit tunnel for the nascent chain.
  • Functional sites: A (aminoacyl), P (peptidyl), and E (exit) sites.
  • GTPase activity: GTP hydrolysis provides energy for factors and translocation during translation.

Wobble Hypothesis

Wobble hypothesis: only the first two nucleotides of a codon (positions 1 and 2) are strictly recognized by the tRNA anticodon; the third base is more flexible, allowing synonymous codons to pair with the same tRNA.

Initiation Phase

General initiation events: Amino acids are attached to their cognate tRNAs by aminoacyl-tRNA synthetases using ATP, producing aminoacyl-tRNA + AMP + PPi. An initiation complex is assembled with the small ribosomal subunit, initiator tRNA, mRNA, and initiation factors; GTP and additional factors facilitate assembly and subunit joining.

Prokaryotic Initiation

Prokaryotes:

  • Initiation codon: AUG, which encodes N-formylmethionine (fMet) for initiation.
  • Met-tRNAf is formylated by methionyl-tRNA formyltransferase using N-formyl-tetrahydrofolate to form N-formylmethionyl-tRNAf.
  • Shine-Dalgarno sequence on the mRNA base-pairs with the 16S rRNA of the small subunit to position the start codon.
  • Initiation factors (IF-1, IF-2, IF-3) facilitate assembly; release of IF-3 allows the large subunit to join and form the full ribosome.
  • Prokaryotic mRNAs are often polycistronic.

Eukaryotic Initiation

Eukaryotes:

  • Initiator amino acid: methionine (Met) is not formylated.
  • Most eukaryotic mRNAs are monocistronic and possess a 5′ cap used for ribosome recruitment.
  • The initiation complex binds the 5′ cap and scans for the start codon; there is no Shine-Dalgarno sequence.
  • Eukaryotic initiation requires many more initiation factors than prokaryotes (around nine or more), making the process more complex.

Differences in Eukaryotes

  1. Transcription is separated from translation in eukaryotes (nucleus vs cytoplasm); in prokaryotes they can occur simultaneously.
  2. Initiator methionine is formylated in prokaryotes but not in eukaryotes.
  3. mRNA recognition for initiation differs (Shine-Dalgarno in prokaryotes vs 5′ cap recognition and scanning in eukaryotes).
  4. Eukaryotic initiation involves many more factors (~9) compared with just a few (3 main IFs) in prokaryotes.
  5. Overall initiation in eukaryotes is more complex; elongation and termination phases are largely conserved between kingdoms.

Elongation Phase

Events during elongation:

  • An aminoacyl-tRNA (aa-tRNA) bound to EF-Tu (in bacteria) plus GTP enters the A site.
  • Peptidyl transferase activity (a ribozyme function of the large subunit rRNA) catalyzes formation of the peptide bond between the peptide on the P-site tRNA and the amino acid on the A-site tRNA.
  • After peptide bond formation, translocation moves the peptidyl-tRNA from the A site to the P site and the deacylated tRNA to the E site; this step requires GTP hydrolysis by translocation factors.
  • The A site is then ready to accept the next aminoacyl-tRNA and the cycle repeats.

Termination Stage

Termination: When a stop codon enters the A site, release factors bind (e.g., RF1, RF2 in bacteria) and additional factors such as RF3 plus GTP promote hydrolysis of the bond between the polypeptide and the tRNA, releasing the completed polypeptide and disassembling the ribosome.

Posttranslational Modifications

Common posttranslational modifications and processing:

  1. Modification of N-termini (e.g., removal of formyl group, signal peptide cleavage)
  2. Proteolytic cleavage of precursor proteins
  3. Side-chain modifications (hydroxylation, methylation, phosphorylation, etc.)
  4. Addition of bulky groups (glycosylation, lipidation, ubiquitination)
  5. Protein folding (often aided by chaperones)

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