Figure 3.

Individual and synergistic activities of eIFs 4A/4B/4G, DHX29, Ded1 and DDX3 in promoting scanning through 5′-UTRs with a highly stable internal stem. (A) Sequence of the 5′-UTR of Stem-2 mRNA. (B–D) Toe-printing analysis of 48S complex formation on (B) Stem-1 and (B–D) Stem-2 mRNAs in the presence of indicated combinations of factors. Initiation codons and the positions of full-length cDNAs and of assembled ribosomal complexes are shown on the sides of each panel. Lanes C/T/A/G depict corresponding DNA sequences. (E) Association of Ded1 (all panels) and DHX29 (left panel) with mammalian 43S complexes (left and right panels) and yeast 40S subunits (central and right panels) was assayed by sucrose density gradient (SDG) centrifugation. Gradient fractions that corresponded to ribosomal peaks were analysed by SDS–PAGE and fluorescent SYPRO staining (left and central panels) or western blotting using anti-Ded1 antibodies (right panel).