FIGURE 8.

The NTD and 7-repeats domains perform overlapping functions in binding to the 40S ribosomal subunit and promoting structural changes in the mRNA entry channel. Purified 40S subunits were incubated with storage buffer, yeIF4B, or yeIF4B variants as indicated in the figure for lanes 0–7 (concentrations listed in Materials and Methods), and the reactions were then incubated −/+Fe²⁺-EDTA, hydrogen peroxide, and ascorbic acid to generate hydroxyl radicals, as detailed in Materials and Methods. The 18S rRNA was extracted from ribosomes incubated with (A,B) WT, ΔNTD, ΔRRM, Δr1-7, ΔNTDΔRRM, and ΔNTDΔRRMΔCTD yeIF4B variants or (C,D) WT and ΔNTDΔr1-7 yeIF4B under each condition and analyzed by primer extension with primer 1371 (A,C) and 1455 (B,D) followed by denaturing gel electrophoresis. Sites of protection from hydroxyl radical cleavage are indicated on the gels.