A multifactor complex of eukaryotic initiation factors, eIF1, eIF2, eIF3, eIF5, and initiator tRNA^Met is an important translation initiation intermediate in vivo

Abstract

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 eIF3 binds to the 40S ribosome and promotes recruitment of the ternary complex; however, physical contact between eIF3 and eIF2 has not been observed. We show that yeast eIF5 can bridge interaction in vitro between eIF3 and eIF2 by binding simultaneously to the amino terminus of eIF3 subunit NIP1 and the amino-terminal half of eIF2␤, dependent on a conserved bipartite motif in the carboxyl terminus of eIF5. Additionally, the amino terminus of NIP1 can bind concurrently to eIF5 and eIF1. These findings suggest the occurrence of an eIF3/eIF1/eIF5/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 eIF5. 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.

Guanine Nucleotide Pool Imbalance Impairs Multiple Steps of Protein Synthesis and Disrupts GCN4 Translational Control in Saccharomyces cerevisiae

“…To confirm that the eIF5-FL Á tRNA i Met association specifically reflects the intracellular levels of MFC that are formed in vivo, isogenic gua1-G388D and GUA1 strains carrying either TIF5-FL or the tif5-7A-FL mutant allele, whose product is defective in forming the MFC (Asano et al 2000), were analyzed as above in strains with wild-type chromosomal TIF5. As expected, no detectable amounts of tRNA i Met were associated with the mutant eIF5-7A-FL protein ( Figure 6B).…”

“…Immunoprecipitation assays: Co-immunoprecipitation assays of Sui3-FL and eIF5-FL proteins were performed using anti-FLAG antibodies as described (Asano et al 1999(Asano et al , 2000. Briefly, cells were cultured in 100 ml of the appropriate medium, harvested at an OD 600 of $0.8, washed, concentrated in 0.6 ml of buffer A (20 mm Tris, pH 7.5; 100 mm KCl; 5 mm MgCl 2 ; 0.1 mm EDTA; 7 mm b-mercaptoethanol, 5 mm NaF; 1 mm phenylmethylsulfonyl fluoride, PMSF) with protease inhibitors and broken with glass beads by three 15-sec pulses in a Fastprep, with 30 sec of cooling between pulses.…”

“…The translation initiation factor 2 (eIF2) bound to GTP recruits the methionyl initiator tRNA (tRNA i Met ) in a ternary complex (TC) and delivers it to the 40S ribosome. This process also involves the formation of an intermediate multi factor complex (MFC) by the addition of other eIFs (eIF1, eIF3, and eIF5) that can exist unbound to 40S in the yeast cells (Asano et al 2000). After the recognition of the initiation AUG codon, GTP hydrolysis promotes tRNA i…”

Starting the protein synthesis machine: eukaryotic translation initiation

“…21 for a current nomenclature of eIF3 subunits). In yeast, a multifactor complex (MFC) of eIF1, eIF2, eIF3, eIF5, and Met-tRNA i Met assembles independently of the ribosome and may be an important functional unit during several stages of translation initiation (22) (Fig. 1).…”

“…1) and structural studies suggest that they form part of an interaction surface with other molecules. (60) Via a simultaneous binding to eIF3c, it is in close proximity to eIF5 and could affect its role as a GTPaseactivating protein (GAP) for eIF2, (22) thus indicating a role for eIF1 in AUG selection.…”