FUNCTIONAL DISSECTION OF EUKARYOTIC INITIATION-FACTOR 4F - THE 4A SUBUNIT AND THE CENTRAL DOMAIN OF THE 4G SUBUNIT ARE SUFFICIENT TO MEDIATE INTERNAL ENTRY OF 43S PREINITIATION COMPLEXES

Eukaryotic translation is initiated following binding of ribosomes eit her to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are bo th mediated by eukaryotic initiation factor 4F (eIF-4F), which consist s of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G; subunit s. Here we present a functional analysis of eIF4F which defines the su bunits and subunit domains necessary for its function in initiation me diated bg the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified transl ation components and lacking eIF4A and -4F, IRES-mediated initiation d id not require the cap-binding protein eIF4E but was absolutely depend ent on eIF4A and the central third of eIF4G. This central domain of eI F4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation condon in all ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES, The central domain of eIF4G was itself UV cross-linked to the IRES and strongly s timulated UV cross-linking of elF4A to the IRES in conjunction with ei ther eIF4B or with the carboxy-terminal third of eIF4G.