EC5: Translation

Question 1

Name the initiation, elongation, and release factors in bacterial and eukaryotic ribosomes.

Answer 1

• Initiation factors
  
  • Bacterial = IF1, IF2, IF3
  • Eukaryotic = many eIFs
• Elongation factors
  
  • Bacterial = EF-Tu, EF-G
  • Eukaryotic = eEF1a, eEF2
• Release factors
  
  • Bacterial = RF1, RF2
  • Eukaryotic, single eRF

Question 2

Polypeptide chains are always made beginning with the same initiator amino acid and a special initiator tRNA. Name them.

Answer 2

Initiation amino acid = methionine (AUG)

Initiation tRNA = tRNA_f

Question 3

Describe the process of polypeptide chain initiation in bacteria.

Answer 3

• 30S ribosomal subunits associate with IF1 and IF3 and are thus prevented from associating with 50S subunits. This keeps 30S subunits free and available for initiation (i.e. ready for mRNA binding)
• IF2 binds the initiator aminoacyl-tRNA and GTP to form met-tRNA_f-IF2-GTP.
• Products of the first two steps bind to mRNA to generate the 30S intitiation complex (30S subunit, mRNA, 3 x IFs, initiator aaRS, GTP).
• The 50S ribosomal subunit joins and triggers GTP hydrolysis which is accompanied by the release of all three IFs. The resultant 70S initiation complex consists of 70S ribosome, mRNA, initiator aaRS.
• The initiation process is complete and the initiator aaRS will recognise the mRNA initiation codon in the ribosomal P site.

Question 4

Name the three bacterial ribosome binding sites for tRNA.

Answer 4

E, P and A

Question 5

Describe the process of polypeptide chain elongation in bacteria.

Answer 5

1. Binding of aminoacyl-tRNA
   Aminoacyl-tRNA enters the ribosomal A sitein response to the mRNA codon downstream of the last one to be decoded. It is carried to the ribosome as a ternary complex with GTP and EF-Tu. During the binding reaction,GTP is hydrolysed and EF-Tu is released bound to GDP. The ribosome now has the initiator aminoacyl-tRNA (or, in subsequent rounds, the nascent peptidyl-tRNA) in the P site and the aminoacyl-tRNA in the A site.
2. Peptide bond formation involves the transfer of the nascent peptide from the tRNA in the ribosomal P site onto aminoacyl-tRNA in the A site. The elongated peptide is now attached to the tRNA in the A site and deacylated tRNA remains in the P site. This reaction is catalysed by the ribosome itself and does not require any other factor.
3. Translocation reaction
   Before the ribosome can accept another aminoacyl-tRNA and make another peptide bond, the mRNA must move through the ribosome so that the next downstream mRNA codon is presented in the ribosomal A site for decoding during the next elongation cycle. Because codons are triplets of nucleotides, such movement must span exactly three mRNA nucleotides to maintain the reading frame. As the mRNA moves, so do the tRNAs. Deacylated tRNA is moved from the P site onto the E site and peptidyl-tRNA moves from the A site onto the P site. The growing peptide chain is advanced along the exit tunnel. This is catalysed by EF-G and involves GTP hydrolysis.

Question 6

Explain the process of polypeptide chain termination.

Answer 6

The RF recognises a stop codon which casues the ribosome to release the newly synthesised polypeptide chain.

The bond is hydrolysed and water acts as a surrogate substrate, so the peptide is released.