title: 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.
authors.affiliation: Ohio State Biochemistry Program Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Devaraj
authors.forename: Kurt
authors.initials: K
authors.lastname: Fredrick
mesh_terms: D000926:Anticodon / Q000235:genetics* / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D003062:Codon; D021901:DNA, Intergenic / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018965:Frameshifting, Ribosomal; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D000926:Anticodon; D003062:Codon; D021901:DNA, Intergenic; D012329:RNA, Bacterial; D012333:RNA, Messenger