In vitro translation initiation assays of WT and mutant IRES RNAs. (A) Diagram of the uncapped monocistronic reporter construct used in these assays. (B) Denaturing gel of each IRES containing reporter construct showing that all RNAs are homogenous in species and concentration. (C) Level of Photinus luciferase after 90 min for WT, ΔdII, and each dIIb mutant. Error bars represent one standard error from the mean of three independent, triplicate experiments. (D) Comparison of the degradation rates of internally radiolabeled WT and dIIb mutant reporter mRNAs in RRL over the course of the experiment. (E) Graph of the degradation rates of WT and dIIb mutant reporter RNAs in RRL over a 90-min time course. Error bars represent one standard deviation from the mean of three experiments.
