SHAPE analysis of ΔdII HCV IRES RNA in the unbound and 40S-bound forms. (A) Representative SHAPE analysis gel of WT full-length and ΔdII HCV IRES RNAs in the free (unbound) form and 40S subunit-bound form. Lanes containing free RNA or bound to the 40S subunit are labeled, reaction lanes are marked as NMIA, control lanes are marked as DMSO (DMSO added, no NMIA) and RT (no DMSO or NMIA added). Lanes 1–4 contain the sequencing reactions. Reference nucleotide numbers are bulleted on the left and the parts of the gel that correspond to different IRES secondary structural elements are indicated by gray bars to the right. The location of the start codon AUG is indicated with red arrowheads. (B) Secondary structure of the ΔdII HCV IRES RNA where decreases in NMIA modification due to 40S subunit binding are designated with gray and regions with increases in modification upon 40S subunit binding are in blue. Structural elements are labeled. The nucleotides 3′ of domain IV (faded gray) are not visible in this analysis, as this is where the primer anneals for reverse-transcription. The part of the ΔdII IRES RNA that shows a different modification than WT full-length IRES, when bound to the 40S subunit, is indicated. (C) Quantitated, normalized, and background-corrected modification data from two independent SHAPE probing experiments, with error bars representing one standard deviation from the mean of both. The experiment was repeated three additional times (data not shown), and the replicates validate the quantitated data shown here. The degree of modification is on the y-axis, each nucleotide is on the x-axis, with the start codon AUG colored red and the location of domain IV indicated. Red bars indicate full-length IRES, blue bars are ΔdII IRES. The top graph contains the modifications for each RNA in the free form; the bottom graph contains a comparison of modifications on each RNA in the 40S subunit-bound form. The dashed box indicates regions that change in their modification pattern when domain II is removed.
