non-standard decoding and ribosomal gymnastics

Question 1

what is a leaky stop codon?

Answer 1

where UAG is present but aa-tRNA recognises the stop codon and translation continues

Question 2

what is a sign of a leaky stop codon in the lab?

Answer 2

pinprick colonies

Question 3

how does a leaky stop codon permit TMV replication?

Answer 3

• plant RNA viruses require a RNA-dependent RNA polymerase ‘replicase’
• 2 products of the replicase gene
• occurs with an efficiency of 2% due to leaky stop codon
• both proteins required

Question 4

how large is the RNA-dependent RNA polymerase ‘replicase’?

Answer 4

126kDa

Question 5

what is programmed frameshifting?

Answer 5

when a stop codon enters the A site of the ribosome, causing it to slip into a different reading frame

Question 6

how does programmed frameshifting produce Release factor 2 in e.coli?

Answer 6

when the UGA stop codon in position 26 reaches the A site, the ribosome slips right. this produces a UUU for aspartic acid in pos 26 rather than a stop codon. the ribosome continues until the stop codon at position 398 is reached

Question 7

give an example of a slippery sequence

Answer 7

the monotonous -A AAA AAG in dnaX gene for e coli

Question 8

give an example of a +1 and -1 programmed frameshift

Answer 8

+1 - release factor 2 in e.coli

-1 - dnaX in e.coli

Question 9

what does the dnaX gene do?

Answer 9

produces two subunits of DNA pol II (71kDa and 47kDa) in equimolar amounts

Question 10

how does the dnaX gene produce two proteins?

Answer 10

contains a slippery monotonous sequence which cause the tRNA to slip when Lysyl-tRNA comes into the A site. this occurs 2/3 across the mRNA, and occurs with 50% efficiency. half read in frame, half read with -1 reading frame