FIGURE 2.

80S assembly and structural probing of the chimeric IGR IRESs. (A) 80S-IGR IRES binding was monitored by competition titration experiments. The fraction of radiolabeled wild-type CrPV IGR IRES bound to 80S ribosomes is plotted against the log of the indicated unlabeled IRES RNA. Representative curves and quantitations are shown from at least three independent experiments. (B,C) Enzymatic probing of the chimeric IGR IRESs. Dicistronic RNAs containing the wild-type and chimeric IGR IRESs were treated with RNase T1 as indicated. Primer extension was performed as described in Materials and Methods using PrEJ69. The reaction products were separated on a denaturing polyacrylamide gel. The nucleotides that are cleaved 3′ by RNase T1 are indicated to the right. A sequencing ladder of the dicistronic construct using the appropriate primer is shown on the left.