Figure 4.

Release of initiation factors from 48S complexes assembled on β-globin mRNA after hydrolysis of eIF2-bound GTP. (A,B) Association of ³²P-eIF3j-(A) and ³²P-eIF3j+ (B) with 48S complexes assembled on β-globin mRNA in the presence of eIF2, eIF3, eIF1, eIF1A, eIF4A, eIF4B, and eIF4F before and after incubation with eIF5, ΔeIF5B, and 60S subunits, as indicated, and assayed after sucrose density gradient centrifugation by Cerenkov counting and gel electrophoresis/autoradiography of peak fractions (inset panels). (C) Association of [³⁵S]Met- with ribosomal complexes formed after incubating 48S complexes with eIF5, ΔeIF5B, and 60S subunits, as indicated, and separated by sucrose density gradient centrifugation. Upper fractions of gradients have been omitted for clarity. Ribosomal complexes are indicated above appropriate peaks. (D,E) Association of eIF1, eIF2, eIF3j- and eIF3j+ with 40S subunits before and after treatment with eIF5 and ΔeIF5B of 48S complexes assembled on β-globin mRNA, analyzed by fluorescent SYPRO staining (D) or immunoblotting (E), after gel electrophoresis of peak fractions obtained after sucrose density gradient centrifugation of ribosomal complexes. eIF2 and eIF3 subunits, eIF1, and ribosomal proteins are indicated. (F) Association of β-globin mRNA with 48S complexes before and after incubation with eIF5, eIF5, and ΔeIF5B, or eIF5, ΔeIF5B, and 60S subunits, and association with 80S ribosomes formed by incubating 48S complexes with eIF5, ΔeIF5B, and 60S subunits, indicated by toeprints 15-17 nt from the initiation codon. The red arrowheads indicate the positions of A, U, and G nucleotides of the β-globin mRNA initiation codon.