Figure 3.
Activities of eIF1 mutants in 48S complex formation. Toeprinting analysis of 48S complexes assembled on β-globin mRNA in the presence of 40S subunits, eIF2, eIF3, eIF4A, eIF4B, eIF4F, eIF1A, aminoacylated initiator tRNA, and the N-terminally truncated eIF1Δ29 mutant (A) or Fe(II)-BABE-derivatized cysteineless and single-cysteine eIF1 mutants (B) as indicated. Reaction mixtures containing only β-globin mRNA are shown in lane 1 of each panel. Reaction mixtures corresponding to lane 2 of each panel contained all translation components except eIF1. Reference lanes A and G depict 18S rRNA sequence generated using the same primer. Complex I is arrested 21–24 nt from the 5′-end of β-globin mRNA. The 48S complex assembled on the AUG triplet is designated as “complex II (+15–17 nt from AUG).” The 48S complex assembled on the GUG triplet located 23 nt from the 5′-end of β-globin mRNA is designated as “+16 nt from GUG.”