Protein Synthesis

Question 1

Describe the two step process of tRNA charging.

Answer 1

1. Specific tRNA synthetase causes reaction between ATP and the cognate amino acid, forming aminoacyl:AMP.
2. Aminoacyl:AMP reacts with cognate tRNA, forming a linkage between amino acid carbonyl and 2’ or 3’ -OH of adenosine.

Question 2

What drives tRNA charging forward?

Answer 2

Hydrolysis of resulting PPi to 2Pi.

Question 3

What are the two domains of aminoacyl tRNA synthetase?

Answer 3

Synthetic: To drive reaction between tRNA and cognate amino acid.
Hydrolytic: Cleaves bond between tRNA and amino acid if it is incorrect (thus increases fidelity of the reaction).

Question 4

Describe translation initiation (5 steps).

Answer 4

1. EIF-2 binds GTP and met:tRNAi.
2. EIF-3/1 complex brings in 40S ribosomal subunit to join initiation complex. Also block premature association of 60S.
3. EIF-4 binds 5’ cap of mRNA and orients it correctly on the ribosome, then scans for first AUG (does so via unwinding secondary structures).
4. 60S ribosomal subunit joins the complex, eIF-3/1 leave, GTP of eIF-2 is hydrolyzed and eIF-2 leaves as well.
5. EIF-4E on 5’ cap and PAB on polyA tail link with eIF-4G scaffold to circularize mRNA to protect from nucleases and ensure only fully processed mRNAs are translated.

Question 5

Describe translation elongation (3 steps).

Answer 5

1. EF-1a brings following charged tRNAs to A site sequentially.
2. Peptides transferase of the ribosomal large subunit transfers amino acid chain from tRNA in P site to single amino acid on tRNA in A site.
3. Decaylated tRNA in P site is displaced to E site, EF-2:GTP hydrolyzes to move peptide holding tRNA from P to A site.

Question 6

Describe translation termination.

Answer 6

1. Stop codon enters A site, release factor eRF1 recognizes this and complexes with eRF3:GTP.
2. Hydrolysis leads to peptide of tRNA in P site being removed, the rest of the complex dissociates.

Question 7

How does initiation of prokaryotic transcription differ from eukaryotic?

Answer 7

Shine Delgado links to a complimentary pyramidine rich sequence on 16S ribosomal subunit to correctly position the mRNA.

Question 8

When and how is eukaryotic translation attenuated at beginning of initiation?

Answer 8

During times of stress.
Kinase phosphorylates eIF-2a. Now it can’t interact with GDP/GTP exchange factor eIF-2b and translation is stopped at initiation.

Question 9

How is eukaryotic translation stopped at end of initiation?

Answer 9

Unphosphorylated 4EBP binds eIF-4e, causing it to not participate in mRNA looping. As such, translation won’t initiate.

Question 10

Describe the diphtheria toxin.

Answer 10

It inactivates EF-2 via ADP ribosylation, stopping its translocase activity so elongation can’t occur.

Question 11

Describe puromycin.

Answer 11

Affects eukaryotes and prokaryotes, and causes premature termination during translation.

Question 12

Describe erythromycin.

Answer 12

Only affects prokaryotes, blocks protein exit tunnel of the 50S ribosomal subunit so protein can’t be released.

Question 13

Describe tetracycline.

Answer 13

Affects prokaryotes only, inhibits binding of following charges aa:tRNA to the A site of the 30 S subunit.

Question 14

Describe streptomycin.

Answer 14

Affects prokaryotes only, causes misreading of mRNA via interaction with S12 of the 30S subunit.

Question 15

Describe chloramphenicol.

Answer 15

Affects prokaryotes and mitochondria, inhibits peptidyl transferase of the 50S subunit.

Question 16

Describe cyclohexamide.

Answer 16

Affects eukaryotes only, inhibits peptidyl transferase of the 60S subunit.