FIGURE 3.

GNRA-dependent RNA–RNA interactions. (A) Secondary structure of the oligoribonucleotides GUAA, UCCG, and GUAG. (B) Mapping of GUAA stem–loop binding site. The GUAA ³²P-labeled oligoribonucleotide (50 nM) was incubated in binding buffer with unlabeled sense IRES domains, antisense, or tRNA (400 nM). Transcripts are named according to the domain they represent; 1–2 (1–85); 3, (84–297); 4–5, (306–462). Transcripts 3ABC (151–227) and stem 3 (84–297, harboring a deletion of residues 152–226) correspond to the apical and basal regions of domain 3, respectively. Complexes were electrophoresed through a 6% acrylamide gel in TBM buffer. Retarded bands corresponding to heterocomplexes are marked with asterisks. (C) Oligoribonucleotides bearing UCCG and GUAG tetraloops modified the efficiency of binding to the entire domain 3 or full-length wild-type FMDV IRES. (D) Substitutions in the GNRA motif of domain 3 alter the efficiency of binding of labeled GUAA stem–loop oligoribonucleotide.