Translation

Question 1

Genetic code is degenerate and non-random

Answer 1

• Genetic code is said to be highly degenerate because more than one mRNA codon may specify a single amino acid
• The arrangement of the code table is non-random

Question 2

Transfer RNA

Answer 2

• Cells utilise “adaptor” molecules called tRNAs that carry a specific amino acid and recognise the corresponding mRNA codon during the process of translation
• Most tRNAS can be schematically arranged in the “cloverleaf” secondary structure with several common features
• tRNAs have numerous post-transcriptionally modified bases that may promote attachment of proper amino acid to the acceptor stem or strengthen codon interactions

Question 3

tRNA Aminoacylation

Answer 3

Accurate translation requires two important recognition steps:

1. The correct amino acid must be selected for attachment to a tRNA by an aminoacyl-tRNA synthetase
2. The correct aminoacyl-tRNA (aa-tRNA) must pair with an mRNA codon at the ribosome

Question 4

How does an aaRS recognise a tRNA so that it can be charged with the correct amino acid?

Answer 4

• Aminoacyl-tRNA synthetases recognise unique structural features of tRNA (e.g. the acceptor stem and anticodon loop are common identity elements for tRNA-aaRS interactions)
• The fidelity of aminoacylation is also thought to be enhanced by proofreading

Question 5

Function of the Ribosome

Answer 5

1. Ribosomes are large complexes of RNA and protein that function as the site of protein synthesis
2. The ribosome binds with mRNA such that its codons can be read with high fidelity
3. The ribosome includes specific binding sites for tRNA molecules
4. The ribosome mediates the interactions of non-ribosomal protein factors that promote polypeptide chain initiation, elongation and termination
5. The ribosome catalyses peptide bond formation
6. The ribosome undergoes movement so that it can translate sequential codons

Question 6

Translation Overview

Answer 6

1. Polypeptide synthesis proceeds from the N-terminus to the C-terminus
2. Chain elongation occurs by linking the growing polypeptide to the incoming tRNA’s amino acid residue
3. Ribosomes read mRNA in the 5’ → 3’ direction
4. Active translation occurs on polysomes: in both bacteria and eukaryotes, multiple ribosomes can bond to a single mRNA transcript, giving rise to a beads-on-a-string structure called the polyribosome

Question 7

Chain Initiation

Answer 7

• Shine-Dalgarno sequence - purine rich (A’s and G’s)
• The tRNA that initiates translation is an unusual form of Met-tRNAMet in which the Met residue is N-formylated
  
  • From hereon, it is referred to as fMet-tRNAfMet
• The process of translation initiation in E. coli requires reh two ribosomal subunits to assemble with fMet-tRNAfMet, on a properly aligned mRNA to form a complex that can commence chain elongation
• This process also requires initiation factors that are not permanently associated with the ribosome
• In E. coli, these are designated IF-1, IF-2 and IF-3
• Initiation results in the formation of an fMet-tRNAfMet.mRNA.ribosome complex in which the fMet-tRNAfMet occupies the ribosome P site while it’s A site is poised to accept an incoming aa-tRNA

Question 8

Chain Elongation

Answer 8

1. Decoding (requires GTP - the ribosome selects and binds an aminoacyl-tRNA whose anticodon is complementary to the mRNA codon in the A site
2. Transpeptidation (peptide bond formation) - the peptidyl group in the P site tRNA is transferred to the aminoacyl group in the A site
3. Translocation (requires GTP) - the A site and P site tRNAs are respectively transferred to the P and E sites, accompanied by their bound mRNA
   
   • That is, the mRNA, together with its base-paired tRNAs, is ‘ratcheted’ through the ribosome by ONE codon

Question 9

Chain Termination

Answer 9

• Release factors (RF-1 or RF-2) recognise the corresponding stop codon in the A site
• The peptidyl-tRNA is hydrolysed and the polypeptide is released, RF-3.GDP binds
• RF-3 exchange its bound GDP for GTP and RF-1 (or RF-2) is released
• RF-3 hydrolyses its bound GTP to GDP+Pi, both of which are released. RRF binds in the A site, followed by EF-GTP
• EF-G hydrolyses its bound GTP to GDP+Pi, which expels the bound tRNAs. RRF, the mRNA and EF-G.GDP depart, causing the ribosomal subunits to dissociate, ready for reinitiation