W7 Prokaryotic Translation

Question 1

Protein synthesis overview:

Answer 1

Initiation: Steps before polypeptide chain formation (Initiation factors)
Elongation: Addition of amino acids to the polypeptide (Elongation factors)
Termination: Stopping of protein synthesis (Release factors)

Question 2

Steps of initiation:

Answer 2

1) mRNA binds to the 30S ribosomal subunit - mediated by the Shine-Dalgani (Ribosome binding site)
2) Binding of an initiator tRNA to the initiator codon (AUG):
-Methionine tRNA (complimentary anticodon to AUG)
-Bound to IF-2 and GTP molecule
-Binds to the P (peptidyl site in the ribosome)
3) Binding of the 50S ribosomal subunit to the 30S initiation complex
-GTP hydrolysis
-Release of IF-1, IF-2, IF-3

Question 3

Additional initiation molecules:

Answer 3

fMet-tRNA:
-Special tRNA (a special methionine)

Question 4

Steps of elongation:

Answer 4

Binding of aminoacyl-tRNA:
1) Aminoacyl-tRNA in complex with EF-Tu and GTP enters the ribosome and binds to the codon in the A site
2) If correct binding occurs between the tRNA and mRNA codon the GTP undergoes hydrolysis and EF-Tu-GDP + Pi is released
3) Discharged EF-Tu is recycled:
-EF-Ts displaces GDP and binds to EF-Tu
-New GTP molecule binds to EF-Tu-Ts
-New aminoacyl-tRNA can then bind to the EF-Tu-GTP complex
Peptide bond formation:
4) Formation of peptide bond between amino acids at P and A sites:
-Bond between amino acid and P site tRNA broken
-Peptide bond between amino acids formed by peptidyl transferase
Translocation:
5) The ribosome translocates one codon distance to the right leaving an empty A site
-EF-G-GTP complex binds to the ribosome
-Hydrolysis of GTP causes the peptidyl-tRNA to move to the P site and uncharged tRNA move to the E site
-EF-G is released and recycled
6) Uncharged tRNA is at E site only transiently
-Prevents aminoacyl-tRNA binding to the A site until translocation is finished
-Uncharged tRNA is then released from the E site
7) Elongation cycle is repeated until a stop codon is encountered
Termination:
8) Stop codon translocates into the A site (No aminoacyl tRNA can bind)
9) RF1/2 (depending on stop codon) binds to the stop codon
10) RF1/2 binding in A site triggers peptidyl transferase to cleave polypeptide from peptidyl tRNA
11) RF3-GDP complex binds to the ribosome causing the release of RF1/2
-GTP replaces GDP on RF3
-Hydrolysis of GTP releases RF3
12) Ribosome recycling factor (RRF) binds to the A site of the ribosome
13) EF-G-GTP binds to ribosome
-Hydrolysis of GTP translocates RRF to the P site and the uncharged tRNA to the E site
14) RRF releases the uncharged tRNA, EG-F releases RRF and the two subunits separate.

Question 5

Bacterial Ribosome (70S):

Answer 5

50S subunit:
23S rRNA + 5S rRNA + 31 proteins

30S subunit:
16S rRNA + 21 proteins