The function of eukaryotic initiation factor 5 (eIF-5) from rabbit reticulocyte lysate has been studied by sucrose gradient preparation of 40 S and 80 S initiation complexes. eIF-5 is required for transfer of initiator tRNA from 40 S preinitiation complexes to puromycin-reactive 80 S complexes. The transfer is dependent upon GTP hydrolysis and is associated with release of eIF-2 and eIF-3 from the 40 S subunit. The GTP-dependent loss of eIF-2 and eIF-3 is catalyzed by eIF-5 in the absence of 60

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... S subunits or when subunit joining is prevented by edeine, but not when GTP is replaced by GuoPP(NH)P. Unstable 40 S subunit . Met-tRNAf complexes generated by eIF-5 can form puromycin-reactive 80 S complexes when 60 S subunits are added in the absence of added GTP. In addition, kinetic evidence is presented that indicates GTP hydrolysis occurs prior to 80 S complex formation.