(A) Relative activity of IRES mutants. Bicistronic constructs of the form CAT-IRES-luciferase carrying the indicated substitutions in motifs A or B were transfected in BHK-21 cells. GUAG and UCCG substitutions in the GNRA motif have been included for comparison. Relative IRES activity was calculated as the luciferase/CAT activity in each extract, made relative to that observed in the wild-type IRES. (B) Enzymatic probing of mutants in motifs A and B. RNA harboring the wild-type sequence or substitution in motifs A or B shown in A was incubated with RNase T2 in native (N) or denaturing (D) buffer and then analyzed by primer extension. Residues marked with asterisks denote new attacks relative to the wild-type sequence; open arrows denote lack of attack in the mutant RNAs.
