Trans-translation and ribosomal peptidyl transferase

Question 1

Bacteria face a major problem during trans-translation when ribosomes..

Answer 1

Become stranded on non-stop mRNA

Question 2

What is non-stop mRNA?

Answer 2

Broken mRNA lacking a stop codon

Question 3

Can you get multiple ribosomes trapped on non-stop mRNAs?

Answer 3

Yes, in polyribosomes

Question 4

What is tmRNA used for?

Answer 4

To release incomplete polypeptides (with C-terminal tags) from ribosomes on non-stop mRNA

Question 5

What does tmRNA resemble?

Answer 5

tRNA lacking an anticodon stem-loop but with an ‘extra’ mRNA-like region (MLR) containing an open reading frame that can be translated

Question 6

What is the ORF in the tmRNA used for?

Answer 6

To put a C-terminal tag on truncated peptide during release from non-stop mRNA

Question 7

What gene encodes the 363 nucleotide E. coli tmRNA?

Answer 7

ssrA gene

Question 8

What tmRNA tag is the target for proteases such as ClpXP?

Answer 8

ANDENYALAA

Question 9

What does tmRNA (ssrA RNA) work together with?

Answer 9

SmpB protein

Forms quaternary complex with EF-Tu + GTP and binds into ribosomal A site

Question 10

Outline the steps that follow when tmRNA-SmpB binds in A site?

Answer 10

• Peptide bond formation
• Translocation brings tmRNA ORF into A site
• ORF of tmRNA is now being translated; aatRNA binds into A site
• Translation of tmRNA ORF generates C-terminal tag on truncated protein; peptide bond formation

Question 11

How many amino acids does the tmRNA ORF encode?

Answer 11

10 amino acid sequence

Question 12

Are ribosomes recycled?

Answer 12

Yes

Question 13

Is the alanine in the tag coded for?

Answer 13

No, it’s inserted into empty decoding site

Question 14

What terminates the synthesis of the tag?

Answer 14

UAA stop codon

Question 15

What does PTC stand for?

Answer 15

(Ribosomal) peptidyl transferase centre

Question 16

What are the CCA-3’ ends of tRNAs in the PTC positioned for?

Answer 16

Peptide bond formation by specific H-bonding to P-loop and A-loops of 23S rRNA

Question 17

What does puromycin resemble?

Answer 17

The 3’ end of aa-tRNA

Question 18

What is puromycin?

Answer 18

An acceptor substrate for peptidyl transferase

Question 19

What happens when the nascent peptide is joined to puromycin?

Answer 19

The peptidyl-puromycin falls off ribosome

Question 20

Is the PTC on the larger or smaller ribosomal unit?

Answer 20

The larger one

Question 21

What do the donor substrates in the P site need?

Answer 21

2’-OH

Question 22

What is the 2’-OH of the P site involved in?

Answer 22

Proton shuttle

Question 23

What are the 3’-CCA ends of tRNAs held in position at the PTC by?

Answer 23

The P-loop and A-loop of 23S rRNA