Protein Metobolism-Biosynthesis

Question 1

Initiation

Answer 1

• 30 s and 50 s subunits make contributions to A and P site
• E site mostly from 50s subuntit
  1) Ribosome binds IF1 (which covers A site) and IF3 (Covers E site on 30 s subunit) and binds the shine delgarno sequence (interacts with 16s rRNA)
  2) IF2-GTP binds (to small 30s subunit) and Fmet-tRNA,fmet (initiating tRNA) comes into the P site
  3) GTP hydrolyzed, all initiation factors leave and 50s subunit comes in

Question 2

Elongation [Loading the tRNA]

Answer 2

1) aminoacyl-tRNA (charged tRNA) binds to Ef-TU-GTP complex
2) The structure formed in step 1 now binds to the A site of the 70s initiation complex
3) GTP hydrolyzed, EF-TU-GDP complex released and charged tRNA sits in A site
4) Tu cycle starts, TS binds to TU-GDP and GDP is kicked out forming (TU-TS).
5) GTP displaces TS thus reforming TU-GTP

Question 3

Elongation [peptide bond formation]

Answer 3

-A to P to E
- 2 molecules of GTP consumed in each round of synthesis (1 from recruiting/1 from translocating)
1) Amino group of A site attacks carbonyl of p site (peptide bond forms)
2) Peptide is now localized on A site tRNA
3) EF-G-GTP (translocation factor) binds to A site causing peptidyl-tRNA (one with peptide) to move over to P site and uncharged tRNA to E site (where it is ejected)
4) GTP hydrolyzed, EF-G-GDP leaves and A site is renewed

Question 4

Termination

Answer 4

1) Release factor binds
2) Peptide cleaved off of tRNA
3) After release factors dissociate, EF-G-GTP and Ribosome Recycling 4) Factor (RRF) replace the release factors
4) EF-G-GTP hydrolyzes and 50s subunit disscoiated from the remaining 30s-tRNA-mRNA
5) IF3 binds to E site on 30s releasing remaining tRNA
6) mRNA falls off, and initiation can begin again

Question 5

Trans-translation steps[form of recycling

Answer 5

• Implemented when ribosome stalls but there is no stop codon
  1) tmRNA (transfer-message tRNA) with protein small protein B (smpB) binds to complex and tmRNA sits in vacant A site
• 5’ end of the tmRNA folds into tRNA shape and the 3’ end acts like mRNA
  2) translation continues but the messed up mRNA and peptide are degraded thus recycling ribosome

Question 6

Charging the amino acid

Answer 6

1) Amino acid is adenylated. C terminus of the amino acid makes a nucleophilic attack on ATP kicking out ppi
2) terminal A then uses (2’ OH or 3’ OH) as a nucleophile on carbonyl carbon (c=o) thus kicking out AMP (loading of amino acid)
3) (dependent on class) if amino acid is on 2’, transesterification happens. 3’ OH is a nucleophile and attacks carbonyl carbon (c=o) (amino acid switches from 2’ to 3’)