Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit c omplex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNA(i) and mRNA. The molecular mechanism by which eIF3 exerts t hese functions is incompletely understood. We report here the cloning and c haracterization of TIF35, the Saccharomyces cerevisiae gene encoding the p3 3 subunit of eIF3. p33 is an essential protein of 30,501 Da that is require d in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p89 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing t he p135, p110, p90, p39, and p38 subunits of eIF3. The p33 subunit binds bo th mRNA and rRNA fragments due to an RNA recognition motif near its C termi nus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA b inding, but expression of the truncated form as the sole source of p33 neve rtheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of pro tein synthesis and that its RNA-binding domain is required for optimal acti vity.