Chemical probing of mutants in the GNRA motif of FMDV IRES. Domain 3 prepared in vitro was annealed to a 5′ end-labeled antisense primer and used as template in a reverse transcriptase reaction. A sequence ladder generated with the same labeled antisense primer was run in parallel to identify extension products; residues are marked on the left as the complementary sense sequence. Lane 1 shows the cDNA products obtained after primer extension of unmodified RNA. Lanes 2 and 3 show primer extension products of the RNA treated with dimethyl sulfate (DMS) in native (N) or denaturing (D) conditions. Filled dots in lane 2 depict the residues that halted RT elongation in DMS-treated RNA, and residues protected from DMS modification are denoted by open circles in lane 3. Transcripts bearing the UCCG or GUAG domain 3 were incubated with DMS in parallel to the wild type. Filled or empty dots point to the residues, marked on the left, which halted RT-elongation in DMS-treated GUAA RNA. Changes observed in GUAG and UCCG mutants are indicated by asterisks (new RT-stop), empty arrow-heads (lack of RT-stop relative to the GUAA RNA), or filled arrows (lack of protection relative to the GUAA RNA), and are listed on the right side of the autoradiogram.
