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

We went on to examine whether the À2 FS product could be engendered by cis-acting stimulatory RNAs and the spacing optima for such events. In these experiments, the AON-binding site was replaced by a pseudoknot (the minimal IBV pseudoknot; a functional version of the wild-type IBV pseudoknot with a shorter loop 3 [12] ), generating plasmid pFSHIV-PK, or a stable stem-loop structure whose base composition was the same as the two stems of the minimal IBV pseudoknot, generating pFSHIV-SL (Figure 7) . A variant of this plasmid with the IBV slippery sequence was also prepared (pFSIBV-SL). Subsequently, spacing variants of these plasmids were constructed (spacers of 3-9 nt) and the mRNAs translated in RRL. With pFSIBV-SL, only the À1 FS product was evident (consistent with the UUUAAAC slippery sequence being incompatible with À2 FS), with efficient À1 FS promoted over a narrow window of spacer length (6-8 nt), peaking at 7 nt. With pFSHIV-SL, both classes of frameshift product were seen. As with AON-mediated frameshifting, À1 FS on the U 6 A slippery sequence was observed at most spacer distances, peaking at 7-8 nt. The À2 FS product was more discrete, spanning spacers of 5-7 nt with a peak at 6-7 nt. Also shown in Figure 7 (panel D) are the translations of pFSHIV-SL-derived mRNA with spacers of 7 or 8 nt in which the first base of the slippery sequence was changed to G, A or C. Similar to the experiment of Figure 4 , we found that disruption of this base inhibited both À1 and À2 FS. With the 8 nt spacer, only a trace of À2 FS is seen, as expected for this spacer length (c.f. Figure 7 panel C), and the inhibition of À1 FS was, if anything, more pronounced than with the 7 nt spacer. To account for the latter, we speculate that with the 7 nt spacer, a greater number of ribosomes have the capacity to frameshift (see Figure 7 panel C) and that partitioning occurs, with ribosomes able to enter either the À1 or À2 frame. With a 7 nt spacer, the block to À2 FS brought about by the slippery sequence changes may well direct 'frameshiftcompetent' ribosomes to partition more into the À1 frame. However, with an 8 nt spacer that does not promote À2 FS (see Figure 7 panel C), there are no additional ribosomes to partition, and the overall level of À1 FS is generally lower. The pattern of frameshifting observed with the stemÀloop stimulator was also seen with the IBV pseudoknot (pFSHIV-PK). Again, À1 FS occurred across a broad range of spacer lengths, peaking at 6-8 nt, with the À2 FS product spanning a more discrete spacer range of 5-8 nt with a peak at 6 nt. Pseudoknot-induced À1 and À2 FS on U 6 A was also examined in constructs containing the SRV-1 gag/ pro pseudoknot, one feature of which is a smaller stem 1 than the IBV pseudoknot (6 bp c.f. 11 bp). As shown in Supplementary Figure S2 , a similar pattern of À1 and À2 FS was observed, with optimal spacing distances of 7-9 nt (À1 FS) and 6-7 nt (À2 FS).