Stem II base pairing contributes to AUG positioning by the IRES. (A) Establishment of primer extension and toeprinting stops for WT IRES in the absence and presence of 40S subunits and comparison to ΔdomII IRES. Primer extension stops were mapped based on dideoxy sequencing reactions. (B) Location of major primer extension stops mapped onto the pseudoknot predicted secondary structure. Primer extension inhibition and toeprinting of (C) the middle and top SII base pair mutants, (D) the bottom SII base pair mutants, and (E) the entire SII base pair mutants. (F) Primer extension reactions of SII mid-5′× IRES-FF luc mRNA (50 nM) in the presence of increasing concentrations of 40S ribosomal subunits. The positions of the major stops are indicated at the right of each gel and are defined in A and B.
