The 'torsional restraint' model predicts that conditions that would inhibit the rotational freedom of the loop region of the pJD366-18-derived mRNA should result in enhanced À1 PRF efficiency. The strategy used in this study was to anneal this mRNA with synthetic oligonucleotides complementary to both the loop region and to the sequence downstream that is normally involved in pseudoknot formation. These 'pseudopseudoknots' would be predicted to restore a pseudoknot-like structure to the mRNA. This is diagrammed in Figure 3A . Two different classes of oligonucleotides having different orientations relative to the mRNA were used to this end: 'joining' (J-) and 'reverse' (R-) oligos. The orientation of the J-oligos promotes the formation of a structure containing the equivalent of a Loop 2 region, while that of the R-oligos promotes a Loop 1 equivalent. The model also predicts that pseudo-pseudoknots having different degrees of rotational freedom should promote different frequencies of ribosome pausing over the slippery site, resulting in different efficiencies of À1 PRF. In order to control this parameter, increasing numbers of nucleotides were inserted between the mRNA hybridizing regions of the J-and R-oligos. The additional non-complementary bases in the J-oligos are 3 0 to the stem-loop residues involved in Stem 2, thus effectively increasing Loop 2. Similarly, the additional non-complementary bases in the R-oligos are 5 0 to the loop acceptor residues and correspond to an increased Loop 1. The structure of the stem-loop of pJD366-18 and its maximum base-paired interactions with representative J-and R-oligos are shown in Figure 3B . To demonstrate that an oligonucleotide-mRNA hybrid was capable of forming under the assay conditions, the J1-oligo was incubated with 5 0 [ 32 P]labeled JD366-18 mRNA and subjected to RNaseH digestion. Digestion of the RNA-DNA hybrid resulted in a labeled 110 nt fragment, demonstrating that the oligonucleotide bound to the mRNA at the position of the pseudoknot (Figure 4) .