Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

Lin, Zhaoru; Gilbert, Robert J. C.; Brierley, Ian

One of the unexpected outcomes of this work was the discovery of À2 FS on the U 6 A heptamer (and potentially-albeit to a lesser extent-on the A 6 C and A 8 C heptamers). Initially, we imagined that the protein in question originated by +1 FS, with the P-site tRNA Phe decoding UUU in the zero frame slipping forwards onto the overlapping+1 frame codon (also UUU) in a proportion of ribosomes stalled at the AON, stem-loop or pseudoknot, but this was ruled out experimentally. A À2 FS is consistent with the idea that mRNA tension promotes 5 0 -movement of the tRNAs in this context. Indeed, the spacing analysis of Figures 6 and 7 provides further support for this viewpoint. Irrespective of the nature of the stimulatory RNA, the spacing distance facilitating maximum À2 FS was consistently $1-2 nt less than that promoting À1 FS. Viewed simplistically, with the shorter spacer, the tension on the mRNA would be greater, increasing the likelihood of a À2 shift. While this hypothesis requires further substantiation, there is a precedent for the importance of mRNA tension, from studies of the +1 programmed frameshifting signal in the E. coli prfB gene encoding release factor 2. In this system, the interaction of a Shine-Dalgarno (SD)-like element in the mRNA with the anti-SD at the 3 0 -end of 16S rRNA is important in promoting efficient +1 FS at a 3 0 -recoding site (52) . The effect of varying the spacing between SD sequence and P-site codon in the prf B system has been analysed by toeprinting (53) . At a spacer length of 2 nt, noticeably shorter than that found naturally between SD and initiator AUG (5 nt; 54), 70S pre-translocation complexes could not be formed and instead, the tRNA added subsequently to fill the A-site moved spontaneously into the P-site, restoring the spacer to the natural length (5 nt). These data support a model in which formation of the SD-anti-SD 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 0 -direction. It is plausible that the À1 and À2 FS we observe here originate in a similar manner, except that here, the tension pulls the mRNA in a 3 0 -direction, leading to À1 and À2 FS.