Translation Initiation in Prokaryotes

Question 1

What are the three steps of translation?

Answer 1

1. Initiation
2. Elongation
3. Termination

Question 2

What is the purpose of initiation in prokaryotic translation?

Answer 2

Ribosome recognizes and assembles on mRNA.

Question 3

What is the purpose of elongation in prokaryotic translation?

Answer 3

Ribosome decodes codons of mRNA in conjunction with aminoacyl-tRNA and synthesizes polypeptide

Question 4

What is the purpose of termination in prokaryotic translation?

Answer 4

Stop codon signals termination

Question 5

What is the first step in prokaryotic translation initiation?

Answer 5

Formation of 30S initiation complex.

Question 6

How does the 30S initiation complex form?

Answer 6

30S subunit binds to the mRNA and places start codon at the P-site
An initiator tRNA binds to the P-site/start codon

Question 7

tWhat does the tRNA that binds to the start codon carrying?

Answer 7

A modified methionine (fMet-tRNA^fMet)

Question 8

What is the second step in prokaryotic translation initiation? What does this consist of?

Answer 8

Formation of 70S initiation complex.
50S subunit binds the 30S initiation complex.

Question 9

How does the prokaryotic 30S subunit bind to mRNA?

Answer 9

Via the ribosome binding site (RBS)

Question 10

What is the consensus RBS sequence?

Answer 10

AGGAGG

Question 11

Where is the RBS located?

Answer 11

8 - 15 nucleotides upstream of start codon, AUG.

Question 12

How does the 16S rRNA of the 30S subunit bind to the RBS?

Answer 12

Via base pairing between RBS sequence and anti-RBS sequence.

Question 13

What is the reverse complement sequence of the RBS?

Answer 13

5’ CCUCCU 3’

Question 14

What does the prokaryotic initiator tRNA do?

Answer 14

Recognizes AUG codon (start codon)

Question 15

How is the initiator tRNA different from regular methionine-carrying tRNA?

Answer 15

CHarged with formylated version of methionine which allows it to bind to the P-site of an incomplete ribosome. Other charged tRNA need to enter the A-site of a complete ribosome.

Question 16

List three proteins required for prokaryotic translation.

Answer 16

Initiation factors
Elongation factors
Release factors

Question 17

Outline the 6 steps of 30S subunit reactivation in prokaryotic translation initiation.

Answer 17

1. IF3 and IF2-GTP bind to inactive 30S subunit
2. IF1 joins the complex, stabilizing it
3. mRNA and tRNAf^Met binds
4. New complex is unstable
5. fMet-tRNA^Met makes contact with start codon in P-site, stabilizing the structure

Question 18

What is important to note about the 30S-IF2-IF3 complex formed in the first step of 30S subunit reactivation?

Answer 18

This complex is relatively unstable.

Question 19

How is it ensured that fMet-tRNA^fMet will bind to the P-site?

Answer 19

IF1 blocks the A-site

Question 20

Outline the remaining steps 7-12 that complete ribosome assembly after 30S subunit reactivation.

Answer 20

7. 50S subunit binds to 30S initiation complex
8. GTP gets hydrolyzed to GDP and Pi
9. GTP hydrolysis causes major conformation change, releasing IF1 and IF3
   10/11/12. IF1 and IF3 release causes further conformational change, resulting in sequential release of Pi and IF2-GDP