SHAPE analysis of dIIb mutants. (A) Model of the placement of domain II and the start codon in the P-site built from the cryo-EM reconstruction of the HCV IRES RNA bound to the 40S subunit (Spahn et al. 2001) (pale transparent yellow and magenta surface), the crystal structure of the 40S subunit from Tetrahymena thermophila (white ribbons) (Rabl et al. 2011), the NMR structure of domain II of the HCV IRES RNA (magenta) (Lukavsky et al. 2003), and the crystal structure of the Thermus thermophilus 70S ribosome with tRNAs and mRNA bound (Selmer et al. 2006) (the mRNA from this structure is shown in blue with the AUG in the P-site in red). This model has little or no steric clash and matches all structural information very well. The model shows the proximity of the tip of domain II (dIIb) to the decoding groove and to the RNA surrounding the AUG. The cryo-EM reconstruction (Spahn et al. 2001) of the full-length IRES (magenta) bound to the 40S subunit (yellow) is inset for reference. DIIb is indicated, and the region where nucleotide G82 is located is indicated by the red box, and the decoding groove and approximate location of the AUG are labeled. (B) Secondary structure cartoon of the HCV IRES with dIIb indicated. Mutants used in this analysis are shown. (C) Representative SHAPE analysis gel of the WT full-length IRES and each of the dIIb mutants. Reactions with or without bound 40S subunit (+/−40S) are in lanes 6–21, the sequencing ladder is in lanes 1–4, and the reverse transcription (RT) control is in lane 5. Reference nucleotide numbers are bulleted on the left and secondary structural domains indicated by gray bars on the right of the gel. The location of the start codon AUG is indicated with red arrowheads. (D) Quantitated, normalized, and background-corrected modification data from two independent SHAPE probing experiments, with the error bars representing one standard deviation from the mean of both. The experiment was repeated two 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 WT IRES, bars in different shades of blue indicate the dIIb mutant IRESs. 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 (relative to WT) when dIIb is mutated.
