All synthetic DNA oligonucleotides were purchased by IDT (Coralville, IA). The modified L-A viral À1 PRF signal containing the G GGU UUA slippery site followed by a simple stem-loop was amplified from pJD18 (23) using the primers luc5 0 b (5 0 -CCCCAAGCTTATGACTTCTAGGCAGGGTTT-AGG-3 0 ) and luc3 0 b (5 0 -CCCCCCATGGGACGTTGTAAA-AACGACGGGATC-3 0 ). These were digested with HindIII and NcoI (restriction sites are underlined) and cloned into the firefly luciferase reporter plasmid pT7-LUC minus 3 0untranslated region-A50 (24) . In the resulting reporter construct (pJD214-18), expression of firefly luciferase requires a À1 frameshift, and the 5 0 sequence of the Stem 2 is not able to base pair with the 3 0 sequence, so that only a stem-loop rather than a pseudoknot is able to form. The same primers were used to amplify DNA from pJDRC (23) to make pJD214-Ry. In this construct, complementary mutations (5 0 -GCUGGC-3 0 to 5 0 -CGACCG-3 0 ) in the 3 0 acceptor sequence of the pseudoknot-forming region of Stem 2 allow the formation of an mRNA pseudoknot that has previously been shown to promote frameshifting at the same frequency as the wild type (23) . The primer luc5 0 CON (5 0 -CCCCAAGCTTATGACTTC-TAGGCAAGGGTTTAGG-3 0 ) contains an additional A nucleotide upstream of the slippery site and was used to make pJD214-0, the zero-frame control. To eliminate the possibility of internal initiation occurring at the luciferase initiation codon downstream of the frameshift signals, the AUG codon was changed to AUA. The Stratagene Quik-Change kit was used to mutate pJD214-18 and pJD214-Ry into pJD336-18 and pJD366-Ry, respectively, using the oligonucleotides 5 0 -GGCGTTCTTCTATGGGACGTTGTA-AAAACGGATC-3 0 and 5 0 -GATCCGTCGTTTTTACAACG-TCCCATAGAAGACGCC-3 0 (the mutated codon is underlined). pJD366-18 was further mutated to make a zero-frame control by placing an A upstream of the slippery site using the oligonucleotides 5 0 -TGACTTCTAGGCAAGGGTTTAGGAG-TG and 5 0 -CACTCCTAAACCCTTGCCTAGAAGTCA (the inserted base is underlined). A series of synthetic DNA oligonucleotides were designed to join the loop acceptor region of mRNA transcribed from pJD366-18 to the downstream region that forms the pseudoknot in the wild-type L-A À1 PRF signal. In the J-oligos, the 3 0 sequences base pair with the loop of the mRNA transcribed from pJD366-18, and the 5 0 regions of these oligos base pair with the downstream sequence. This orientation is reversed for the R-oligos. These general orientations are shown in Figure 3B . The naming of the oligonucleotide names refers to the number of additional residues placed between the regions of complementarity. The bases complementary to the pJD366-18 sequence are underlined.