Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

Abstract

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

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

“…The accumulation of eIF1A on 48S complexes in the fun12-td strain (Fig. 1C) combined with the results of prior studies revealing a direct interaction between the C termini of eIF5B and eIF1A (9,24,28) led us to test the functional importance of the eIF5B-eIF1A interaction. Replacing the last five residues of eIF1A (residues 149 to 153, encoding DIDDI) with alanine (the mutant named eIF1A-5A) resulted in a slow-growth (Slg Ϫ ) phenotype on synthetic complete (SC) medium containing all amino acids ( Fig.…”

“…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).…”

Starting the protein synthesis machine: eukaryotic translation initiation

“…(64) Domain IV is essential for eIF5B function and interacts with eIF1A, suggesting that the release of eIF1A and eIF5B from the ribosome could be coupled. (57) Thus, formation of an elongationcompetent 80S ribosome requires two distinct GTP hydrolysis steps, which predominantly serve as checkpoints for proper AUG codon identification and 80S assembly.…”

“…eIF1A has also been implicated in other aspects of translation initiation. It was shown to promote binding of the ternary complex to the 40S subunit, (56) it binds to eIF5B (the homologue of bacterial IF 2), (57) primarily through their CTDs, and the NTD of eIF1A binds to eIF2 and eIF3. (58) The NMR solution structure of eIF1A shows a modular organisation with an unstructured, basic NTD, a central five-stranded b-barrel oligonucleotidebinding (OB) domain, responsible for sequence-independent binding to single-stranded RNA, and an additional acidic CTD comprising two a-helices followed by an unstructured tail.…”