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Short spacing between the Shine–Dalgarno sequence and P codon destabilizes codon–anticodon pairing in the P site to promote +1 programmed frameshifting
Author(s) -
Devaraj Aishwarya,
Fredrick Kurt
Publication year - 2010
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2010.07421.x
Subject(s) - shine dalgarno sequence , biology , transfer rna , translational frameshift , genetics , ribosome , p site , start codon , release factor , messenger rna , stop codon , a site , microbiology and biotechnology , binding site , gene , rna
Summary 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.