SHAPE analysis of wild-type HCV IRES RNA in the unbound and 40S-bound forms. (A) Schematic of HCV IRES translation initiation where the IRES first binds the 40S subunit (orange), then eukaryotic initiation factor (eIF) 3 (green), and the ternary complex (eIF2-GTP-Met-tRNAiMet, blue), followed by GTP hydrolysis and eIF release. The 60S subunit (red) then joins to form an 80S ribosome. Removal of domain II (ΔdII) blocks the formation of 80S ribosomes. (B) Representative SHAPE analysis gel of full-length HCV IRES RNA in the free form (left) and 40S subunit-bound form (right). Lanes containing free RNA or bound to the 40S subunit are labeled, reactions lanes are marked as NMIA (two concentrations of NMIA are shown), control lanes are marked as DMSO (DMSO added, no NMIA) and RT (no DMSO or NMIA added). Lanes 1–4,9–12 are 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. (C) Secondary structure of the full-length HCV IRES RNA, where decreases in NMIA modification upon 40S subunit binding are designated with gray, and regions with increases in modification 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. (D) 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 experiments. 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 free IRES; gray bars are 40S bound IRES. Gray regions indicate a decrease in modification upon 40S subunit binding and cyan regions indicate an increase in SHAPE modification upon the addition of the 40S subunit.
