Though all of the cited studies support the torsional restraint model, none has directly addressed it. In the experiments presented in this study, a series of 'pseudo-pseudoknot' containing reporter constructs were used to test the torsional restraint hypothesis. In vitro frameshifting assays show that frameshifting can be significantly stimulated by limiting the rotational freedom of the loop region of a stem-loop structure, and that the degree of rotational freedom of Stem 1 is important in determining the extent of À1 PRF. Furthermore, mRNA toeprint analyses reveal a pseudo-pseudoknot-specific strong stop 16 nt 3 0 of the slippery site, consistent with this structure being able to direct ribosomes to pause with their A-and P-sites positioned at the slippery site.