Investigation of possible base-pairing between domain IIb and domain IV. (A) Comparison of the sequences at the apical loop of domain II (dIIb) and the RNA in and around the AUG start codon (underlined) for 11 IRES RNAs proposed to have HCV-like secondary structures (Pestova and Hellen 1999; Hellen and de Breyne 2007). Boxes around parts of each sequence indicate the proposed location of the dIIb apical loop. Sequences were aligned based on the proposed position in each secondary structure. The possible base-pairing interaction in the HCV IRES is indicated with bars above the aligned sequences. In each sequence, red indicates stretches of two or more bases that could potentially pair (identified by visual examination). Uppercase red letters could form Watson–Crick pairs; lowercase could form G•U pairs. The pairing sequences, number of potential pairs, and location of these pairs is not conserved. (B) Model from Figure 3A with only the tip of domain II and the position of the mRNA and AUG in the P-site shown. The location of bases that could pair is shown with various colors; pairing partners have matching colors. The distance between potential pairing bases is shown. The cryo-EM reconstruction (Spahn et al. 2001) of the full-length IRES (magenta) bound to the 40S subunit (yellow) is inset for reference. The part of the cryo-EM shown in the model is boxed in red. (C) Cartoon of HCV IRES secondary structure with mutations made in dIIb and dIV highlighted in red, and the putative base-pairing interaction indicated by lines connecting the two domains and yellow shading. AUG is indicated at the apex of dIV. (D) Quantitated, normalized, and background-corrected modification data from two independent SHAPE probing experiments of the WT IRES RNA and the mutants shown in C, all bound to the 40S subunit, with error bars representing one standard deviation from the mean of both. Note that the additional repeat for both the unbound and the IRES•40S bound state (data not shown) validates 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. Orange bars indicate WT IRES; bars in different shades of purple indicate mutants. The top graph contains the modifications for each RNA in the dIIb apical loop region; the bottom graph contains a comparison of modifications on each RNA in the dIV region. The start codon AUG is indicated in red.
