Translation 2

Question 1

Where do IF-1 and IF-3 bind in prokaryotic initiation?

Answer 1

The small 30S subunit. IF-1 binds to the A site.

Question 2

What does IF-3 binding do?

Answer 2

Prevents premature assembly of the 30S subunit with the 50S subunit.

Question 3

How does mRNA bind to the 30S subunit?

Answer 3

By using the Shine Dalgarno sequence.

Question 4

Where is the Shine Dalgarno sequence found?

Answer 4

8 bases upstream of AUG.

Question 5

How is the first Met-charged tRNA brought into the ribsome?

Answer 5

Brought into the P site on the small subunit with IF2-GTP.

Question 6

What does GTP hydrolysis drive during initiation?

Answer 6

A major conformational change that allows the large subunit and the small subunit to join. All initiation factors are then released.

Question 7

What are the differences between eukaryotic and prokaryotic translation initiation?

Answer 7

• Eukaryotes require more initiation factors.

- Eukaryotes don’t have a Shine Dalgarno sequence.

Question 8

How is the ribosome positioned in eukaryotic translation?

Answer 8

By interactions with both ends of the mRNA- the 5’cap and the poly(A) tail.

Question 9

How is the second charged tRNA added?

Answer 9

It is brought into the A site by EF-TuGTP. GTP hydrolysis causes a conformational change, locking the tRNA in place and releases EF-TuGDP.

Question 10

How is EF-TuGTP regenerated during translation elongation?

Answer 10

Regenerated by EF-Ts, a guanine exchange factor.

Question 11

How is peptide bond formation catalysed during elongation?

Answer 11

Catalysed by 23S rRNA. Occurs via nucleophilic attack of the C=O group of the peptide in the P site, by the NH2 group of the amino acid in the A site.

Question 12

How is the ribosome moved along to the next codon after peptide bond formation?

Answer 12

Requires EF-G and GTP hydrolysis. Moves the uncharged tRNA into the E site and the peptidyl-tRNA into the P site, leaving the A site available for the next charged tRNA.

Question 13

Compare translation elongation in prokaryotes and eukaryotes.

Answer 13

Occurs via the same mechanism. Eukaryotes have analogous elongation factors; eEF1α, eEF1βγ and eEF2.

Question 14

How is translation terminated?

Answer 14

A release factor binds to the A site and the ester bond between the C=O of the amino acid and the OH of the tRNA is hydrolysed. All of the ribosome subunits, uncharged tRNA and release factors dissociate.

Question 15

Compare translation termination in prokaryotes and eukaryotes.

Answer 15

In prokaryotes there are different release factors to recognise different stop codons- RF1 for UAA, RF2 for UAA and UGA.
In eukaryotes there is only one release factor- eRF.

Question 16

How can translation be inhibited?

Answer 16

By antibiotics that mimic translation factors, e.g. Tetracyclins block the A site.

Question 17

What suggests that there are structural differences between the eukaryotic and prokaryotic ribosomes?

Answer 17

There are antibiotics that specifically inhibit only one. For example, chloramphenicol blocks peptidyl-transferase but cycloheximide only blocks the eukaryotic peptidyl-transferase.

Question 18

How does EF-Tu improve translation accuracy?

Answer 18

As it binds the charged tRNA, it checks that the amino acid and the tRNA are correctly matched. Charged tRNAs are in a bent conformation when bound to EF-TuGTP:

• allows codon pairing, but not addition of the amino acid to the peptide
• GTP hydrolysis will only occur if the codon and anticodon are correctly matched.

Question 19

What is the level of translation accuracy in a ribosome?

Answer 19

99.99% accuracy.

Question 20

What happens to incorrectly matched tRNAs?

Answer 20

They dissociate quicker than correctly matched tRNAs due to weaker binding to the codon and are not used in protein synthesis.

Question 21

Why can EF-G bind the A site of the ribosome?

Answer 21

The EF-G protein, also called translocase, can bind the A site of the ribosome because its structure resembles the EF-Tu/tRNA complex.

Question 22

Why is the EF-G (translocase) protein needed for movement of the ribosome along the mRNA?

Answer 22

In binding the A site, EF-G protein displaces the peptidyl-tRNA bound there.