FIGURE 3.

Translational activities of the chimeric IGR IRESs. (A) Uncapped dicistronic RNAs containing wild-type or chimeric IGR IRESs were incubated in RRL at 30°C for 60 min in the presence of [³⁵S]methionine. The first cistron, encoding Renilla luciferase (Rluc), measures scanning-mediated translation, and the second cistron, firefly luciferase (Fluc), measures IGR IRES-mediated translation. Shown is a representative gel of radiolabeled Fluc and Rluc protein products detected by autoradiography. Where applicable, the amount of the ternary complex inhibitor NSC119889 added to the reactions is shown above the gel. (B) Quantitations of translational activities of the chimeric IRESs. The ratios of firefly to Renilla luciferase are shown and are normalized to the ratio of the dicistronic RNA containing the wild-type CrPV IGR IRES. (C) Normalized quantitation of the chimeric IRES translation under NSC119889 treatment. Firefly (top) and Renilla (bottom) luciferase activities were normalized to the translational activity of each dicistronic RNA in the absence of NSC119889. The data shown are the averages of at least three independent experiments ±SD. (D) Translational activities of the chimeric IRESs in the presence of edeine. Shown is a representative gel of radiolabeled Fluc and Rluc detected by autoradiography. The bottom panel shows quantitations of Rluc and Fluc, normalized to the amount of luciferase produced by each dicistronic RNA in the absence of edeine. The average value from three independent experiments ±SD is shown.