The IRES helical stem tolerates bulges and a 1-basepair deletion.

<p> (A) Schematic of the IRES PKI helical stem is shown, with the region bearing mutations highlighted in grey. The respective mutations resulting in a 1-nucleotide bulge (ΔU₆₁₇₆), 2-nucleotide bulge (ΔUU_6176-7), 1-basepair deletion (ΔU₆₁₇₆/A₆₂₀₀) and 2-basepair deletion (ΔUU_6176-7/AA_6199-200) are shown. (B) Translational activities of mutants bearing alterations in the PKI helical stem. Uncapped dicistronic reporter RNAs containing the wild-type or mutant IGR IRES in the intercistronic space were incubated in RRL in the presence of [³⁵S]-methionine. Shown are the average values ± S.D. for the ratios of FLuc to RLuc expression, normalized to the wild-type IGR IRES from at least three independent experiments. (C) Toeprinting analysis for IRESs bearing mutations in the helical stem. Dicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in the absence [−] or presence [+] of purified salt-washed HeLa 80S (100 nM). Primer extension analysis was performed using PrEJ69 in the presence of α-[³²P]-dATP. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The sequencing reaction for the wild-type IRES is shown on the <i>left</i>, with the respective nucleotide numbers as indicated. The locations of the major toeprints are as indicated on the <i>right</i>. (D) Summary of the translational activities and toeprint intensities for the mutant IRESs. Toeprint intensities were measured as a fraction of the radioactive counts for Toeprint A over the total radioactive counts within each lane, normalized to that of the wild-type IGR IRES.