Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

The initiation of protein synthesis in eukaryotes is a complex series of events orchestrated by initiation factors (eIFs). In vitro studies using purified translation initiation factors have led to the elucidation of the translation initiation pathway in which the different factors associate with the 40S ribosomal subunit and guide the binding of Met-and mRNA. While the essential in vitro functions of the different factors are well established, the precise timing of factor binding to and release from the ribosome is less well understood. Moreover, not all of the in vitro-defined functions of the factors have been confirmed in vivo. The first step in translation initiation involves the binding of factor eIF2 to GTP and Met-forming a ternary complex. The ternary complex along with factors eIF1, eIF1A, eIF3, and eIF5 binds to the 40S ribosome, forming the 43S complex (reviewed in reference 16). The 43S complex then binds to an mRNA near the 5′ cap, forming a 48S complex that scans along the mRNA in a 3′ direction in search of an AUG start codon. In the 48S complex, some of the eIF2-GTP is hydrolyzed to eIF2-GDP + Pi (2). Upon AUG codon recognition, GTP hydrolysis is completed, and Pi is released, converting the 48S complex from an open to a closed complex with Met-in the P site of the 40S subunit (29). The release of Pi from the 48S complex appears to be coupled with displacement of eIF1 from its binding site near the P site (2, 22). In addition, the majority of eIF2 either dissociates from or repositions on the 48S complex following AUG codon recognition (32). Intriguingly, factors eIF3, eIF1, and eIF1A appear to be retained on the 48S complex following eIF2 release (38). Conversion of the 48S complex to a functional 80S ribosome requires joining of the large 60S ribosomal subunit in a reaction catalyzed by factor eIF5B (30). In the yeast Saccharomyces cerevisiae, eIF5B is encoded by the nonessential FUN12 gene. Cells lacking eIF5B display an extremely slow-growth phenotype, an altered polysome profile consistent with a defect in translation initiation, and are unable to grow under amino acid starvation conditions due to the failure to derepress translation of the GCN4 mRNA (8). While in vitro reconstitution experiments have demonstrated the role for eIF5B in promoting subunit joining (3, 30, 36), in vivo data supporting this precise function have not been reported. As eIF3 and eIF1 are not present on the 80S complex following subunit joining (38), it is thought that these factors dissociate during subunit joining. eIF1A is a eukaryotic ortholog of the bacterial translation initiation factor IF1, which binds in the ribosomal A site, and eIF5B is an ortholog of the factor IF2. Like bacterial IF1, eIF1A possesses a conserved β-barrel fold (5, 34); however, the eukaryotic factor has in addition a helical element that packs against the core domain and the N- and C-terminal nonstructured tails. eIF1A functionally coordinates with eIF1 and promotes 43S complex formation, as well as ribosomal scanning and AUG start codon recognition (12, 23, 29). In addition to these early roles in the translation pathway, eIF1A physically and functionally interacts with eIF5B (9, 24, 28). The C terminus of eIF1A binds to eIF5B, and structural analysis revealed that the eIF1A C-terminal residues pack into a groove formed by helices H13 and H14 in C-terminal domain IV of eIF5B (24). This eIF5B-eIF1A interaction appears to be important for protein synthesis. First, overexpression of eIF1A exacerbates the slow-growth phenotype in strains lacking eIF5B (9). Second, deletion of or substitution of Ala for the five C-terminal residues (DIDDI) in yeast eIF1A impairs 80S complex formation and eIF5B ribosome-dependent GTPase activity in vitro (1). Third, deletion of helix H14 impairs eIF5B GTPase and ribosome-joining activities (1). As eIF1A binds to the 40S subunit early in the translation initiation pathway, it has been proposed that the eIF5B-eIF1A interaction helps recruit eIF5B to the 48S complex. Following subunit joining, GTP hydrolysis by eIF5B triggers a switch enabling release of the factor from the 80S ribosome (36). The timing of eIF1A release from the ribosome has not been examined, and it is unknown whether the eIF5B-eIF1A interaction enables the release of eIF1A in concert with eIF5B. In this report we utilize a degron approach in which eIF5B is tagged with an unstable ubiquitin fusion protein to rapidly deplete eIF5B in yeast cells (11, 18). Analysis of translation initiation complexes from these cells revealed the accumulation of 48S complexes, providing in vivo evidence that eIF5B promotes ribosomal subunit joining. Consistent with a direct interaction between eIF5B and eIF1A, C-terminal mutations in these factors impaired the ability of eIF1A to recruit eIF5B to the 48S complex. Finally, blocking GTP hydrolysis by eIF5B led to the accumulation of both eIF1A and eIF5B on the 80S products of subunit joining both in vivo and in vitro. Based on these findings, we propose a model in which eIF1A helps recruit eIF5B to the 48S complex to promote subunit joining and that the subsequent release of eIF1A is dependent on GTP hydrolysis and the release of eIF5B from the 80S ribosome.