Stem–loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

Yu, Chien-Hung; Noteborn, Mathieu H.; Pleij, Cornelis W. A.; Olsthoorn, René C. L.

In contrast to earlier reports involving the IBV frameshifting pseudoknot (21, 22) , we found that in the case of the SRV-1 gag-pro frameshift inducing pseudoknot a hairpin of similar composition as the pseudoknot did stimulate frameshifting in vitro ( Figure 1A and B). The 12 bp hairpin derivative of the SRV-1 pseudoknot (SRV-hp) showed 22% frameshifting efficiency, whereas the SRV-1 pseudoknot (SRV-pk) in this context yielded 31%. The pseudoknot in these experiments is a modified version of the wild-type SRV-1 pseudoknot previously used for NMR and functional analysis (14) . We note that the U UUAAAC slippery sequence was used to enhance the sensitivity of the in vitro frameshifting assay. This sequence is $1.5-fold more slippery than the wild-type GGGAAAC slippery sequence (28) . In the latter context, the hairpin was indeed less efficient (data not shown) while a non-slippery variant, GGGAAGC, was not effective at all (<0.2%, data not shown). Two other known efficient slip sites, AAAAAAC and UUUUUUA, caused 23 and 27%, respectively, of ribosomes to switch frame in the presence of the 12 bp hairpin (data not shown). These data showed that the 12 bp hairpin is a genuine stimulator of frameshifting. Since the hairpin construct also contained sequences resembling those of L2 of the pseudoknot construct, it was theoretically possible that these nucleotides could take part in the same base triples. To investigate this possibility, we replaced the downstream sequence in the hairpin construct (SRV-muthp). This did not affect the frameshift efficiency of the hairpin construct. In contrast, the same mutations in the pseudoknot context (SRVmutpk) reduced its activity about 6-fold ( Figure 1B) . Thus, it is unlikely that triple helix formation or other tertiary interactions contribute to hairpin-dependent frameshifting; the hairpin as such seems to be sufficient.