Short spacing between the Shine-Dalgarno sequence and P codon destabilizes codon-anticodon pairing in the P site to promote +1 programmed frameshifting.

Abstract:

:Programmed frameshifting in the RF2 gene (prfB) involves an intragenic Shine-Dalgarno (SD) sequence. To investigate the role of SD-ASD pairing in the mechanism of frameshifting, we have analysed the effect of spacing between the SD sequence and P codon on P-site tRNA binding and RF2-dependent termination. When the spacing between an extended SD sequence and the P codon is decreased from 4 to 1 nucleotide (nt), the dissociation rate (k(off) ) for P-site tRNA increases by > 100-fold. Toeprinting analysis shows that pre-translocation complexes cannot be formed when the spacer sequence is ≤ 2 nt. Instead, the tRNA added secondarily to fill the A site and its corresponding codon move spontaneously into the P site, resulting in a complex with a 3 nt longer spacer between the SD-ASD helix and the P codon. While close proximity of the SD clearly destabilizes P-site tRNA, RF2-dependent termination and EF-Tu-dependent decoding are largely unaffected in analogous complexes. These data support a model in which formation of the SD-ASD helix in ribosomes stalled at the in-frame UGA codon of prfB generates tension on the mRNA that destabilizes codon-anticodon pairing in the P site and promotes slippage of the mRNA in the 5' direction.

journal_name

Mol Microbiol

journal_title

Molecular microbiology

authors

Devaraj A,Fredrick K

doi

10.1111/j.1365-2958.2010.07421.x

subject

Has Abstract

pub_date

2010-12-01 00:00:00

pages

1500-9

issue

6

eissn

0950-382X

issn

1365-2958

journal_volume

78

pub_type

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