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. Author manuscript; available in PMC: 2014 Jul 22.
Published in final edited form as: Nature. 2013 Nov 3;503(7477):539–543. doi: 10.1038/nature12658

Figure 4. Binding of eIF3 to subdomain IIIb of the CSFV IRES and effects on translation of the eIF3/IRES interaction.

Figure 4

(a) eIF3 binding site on the CSFV IRES. The residues potentially interacting with eIF3 from domain IIIb in the CSFV IRES are highlighted by blue spheres and labeled (bottom). (b) Toeprinting analysis of 48S complexes assembled on wild-type HCV (nt. 1–349)–CAT mRNA (“wt HCV IRES”) and ΔIIIb-HCV IRES or ΔIIIc-HCV IRES derivatives22 lacking either domain IIIb (nt. 172–227) or domain IIIc (nt. 229–238) with 40S subunits, Met-tRNAiMet, eIF2 and eIF3 as indicated. Primer extension was arrested at nts. 342–345 by stably bound 40S subunits2 and at nts. 355–359 by 48S complexes, as indicated. Lanes C, T, A and G show the cDNA sequence corresponding to wt HCV IRES mRNA. The position of the initiation codon AUG373 is indicated on the left. (c) Inhibition of 43S complex formation by subdomain IIIabc of HCV and CSFV IRESs, assayed by sucrose density gradient centrifugation (SDG). The protein composition of ribosomal peak fractions was analyzed by SDS-PAGE and fluorescent SYPRO staining. (d) Inhibition of 48S complex formation on β-globin mRNA by IIIabc subdomains and by complete HCV and CSFV IRESs containing a 4nt. deletion in helix III2, assayed by toe-printing. Lanes C, T, A and G show the cDNA sequence corresponding to β-globin mRNA. The position of the initiation codon is indicated on the left. Each gel reported in the figure is representative of results obtained from three technical replicates.