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.

Most RNA viruses that make use of ribosomal frameshifting employ pseudoknot structures instead of simple hairpins for this job. The reason for this may be the presence of a triple helix interaction between S1 and L2 in most frameshifter pseudoknots, which has been suggested to be a poor substrate for the ribosomal helicase (13, 33) and hence increases ribosomal pausing and the time window for slippage. Although pausing is critical, it is not sufficient for efficient frameshifting (34) . Previously, it was shown that a 17 bp hairpin with a calculated stability of À31.2 kcal/mol derived from the minimal IBV pseudoknot induced 5-to 10-fold less frameshifting in RRL (22) than its parent pseudoknot even though both the hairpin and the pseudoknot can pause ribosomes at the same position and to a similar extent (34) . In the present study, a 12 bp hairpin derivative of the SRV-1 gag-pro pseudoknot with a calculated stability of À26.9 kcal/mol was capable of inducing 22% of frameshifting, which is only 1.4-fold . Influence of loop sequence and closing base pair (cbp) on À1 ribosomal frameshifting efficiency. The composition of various loops capping a 9 bp stem is shown in bold, and CG-cbps are shown in lower case. The constructs are named after their loop sequence followed by the '/cg' extension when the cbp was changed from G-C to C-G. Slippery sequence and spacer are the same as in the construct shown in Figure 1A . Graph is similar to that of Figure 2B except that on the right y-axis ÁG starts from À18 kcal/mol. less than its pseudoknotted counterpart. This indicated that a non-natural hairpin can be an efficient frameshift stimulator, at least in the SRV-1 model. Furthermore, our results showed that the frameshifting efficiency increased upon elongation of the length of the hairpin up to 12-15 bp, which is consistent with our previous data using antisense oligonucleotides of 12-15 nt to induce ribosomal frameshifting (35) . More importantly, the frameshift inducing ability of these hairpin constructs with a perfect stem linearly correlated with the calculated thermodynamic stability, in agreement with two previous reports (19, 20) .