Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4Astrongly enhances binding of elF4G to the internal ribosomal entry site ofencephalomyocarditis virus and is required for internal initiation of translation

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into thr ee similarly sized regions. The central region (amino acids [aa] 613 to 109 0) binds eIF3, eIF1A, and the encephalomyocarditis virus (EMCV) internal ri bosomal entry site (IRES) and mediates initiation on this RNA. We identifie d the regions of eIF4GI that are responsible for its specific interaction w ith the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recog nition motif (RRM)-like domain. An additional amino-terminal sequence (aa 7 22 to 746) was required for binding eIF4A and for 48S complex formation. eI F4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not be ta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex forma tion even if they bound the IRES normally. These data indicate that the eIF 4G-eIF4A complex, rather than eIF4G alone, is required for specific high-af finity binding to the EMCV IRES and for internal ribosomal entry on this RN A.