A multifactor complex of eukaryotic initiation factors, eIE1, eIF2, eIF3, eIF5, and initiator tRNAMet is an important translation initiation intermediate in vivo

Translation initiation factor 2 (eIF2) bound to GTP transfers the initiator methionyl tRNA to the 40S ribosomal subunit. The eIF5 stimulates GTP hydrolysis by the eIF2/GTP/Met-tRNA(i)(Met) ternary complex on base-pairing between Met-tRNA(i)(Met) and the start codon. The eIF2, eIF5, and eIF1 all have been implicated in stringent selection of AUG as the start codon. The eIE3 binds to the 40S ribosome and promotes recruitment of the ternary complex; however, physical contact between eIE3 and eIE2 has not been observed. We show that yeast eIE5 can bridge interaction in vitro between eIE3 and eIF2 by binding simultaneously to the amino terminus of eIF3 subunit NIP1 and the amino-terminal half of eIE2 beta, dependent on a conserved bipartite motif in the carboxyl terminus of eIF5. Additionally, the amino terminus of NIP1 can bind concurrently to eIF5 and eIE1. These findings suggest the occurrence of an eIE3/eIE1/eIE5/eIF2 multifactor complex, which was observed in cell extracts free of 40S ribosomes and found to contain stoichiometric amounts of tRNA(i)(Met). The multifactor complex was disrupted by the tif5-7A mutation in the bipartite motif of eIE5. Importantly, the tif5-7A mutant is temperature sensitive and displayed a substantial reduction in translation initiation at the restrictive temperature. We propose that the multifactor complex is an important intermediate in translation initiation in vivo.