Having demonstrated the utility of the in vitro frameshifting assay and that the J-and R-series of oligonucleotides were able to hybridize with synthetic mRNA produced from pJD366-18, the next step was to monitor frameshifting efficiencies promoted by these hybrid species. Significant increases in frameshifting were observed with the incubation of pJD366-18 mRNA with oligonucleotides J1 ($10%) and J2 ($35%), while only modest increases were seen with J3 and J4 ( Figure 5 ). These findings are consistent with the notion that changes in the degree of rotational freedom of the structure would affect the distribution of paused ribosomes in the vicinity of the slippery site. One potential complication with the J-oligos is the possibility that they could interact with the Loop 2-Stem 1 region. In the R-oligos, the additional bases are distal to any possible Loop 2-Stem 1 interactions and would be more analogous to increasing Loop 1. The R-oligos stimulated À1 PRF to an even higher extent than the J-oligos ( Figure 5 ). Importantly, increasing the length of the bridging regions in these oligonucleotides (R1 to R3), which is predicted to increase the rotational freedom of the stem-loop, resulted in decreased frameshifting activity as predicted by the torsional resistance model. However, addition of three residues between the two binding regions of the R-oligo (R4) resulted in an unexpected increase in frameshifting with a very large amount of variation.