The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the ␥ subunit of the heterotrimeric eIF2; however, only eIF2␤, and not eIF2␥, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2␥, as well as its isolated G domain, binds directly to eIF5 and the ⑀ subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50 -100 of yeast eIF5 impairs the interaction with recombinant eIF2␥-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2␥-G domain function via eIF2␤, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2␥-G domain.The initiation of protein synthesis in eukaryotic cells requires the coordinated activity of at least 10 eukaryotic initiation factors (eIFs), 2 with several of the factors composed of multiple polypeptide chains. Interactions among the initiation factors and the ribosome promote initiator methionyl-tRNA (Met-tRNA i Met ) and mRNA binding to the 40 S ribosomal subunit and subsequent joining of the large 60 S ribosomal subunit to form the translationally competent 80 S ribosome. The factor eIF2 is responsible for delivering Met-tRNA i Met to the 40 S subunit in the first step of translation initiation (reviewed in Refs. 1 and 2). The eIF2 is composed of three polypeptide chains: the core eIF2␥ subunit (GCD11 in yeast), to which the eIF2␣ (SUI2) and eIF2␤ (SUI3) subunits bind. The eIF2␥ subunit contains a consensus GTP-binding (G) domain at its N terminus, and structural studies on the eIF2␥ homolog from archaea revealed a three-domain structure that closely resembles the structure of the translation elongation factor EF-Tu (EF1A) (3, 4). The structural similarity between eIF2␥ and EF-Tu is consistent with the common function of the two proteins to bind aminoacyl-tRNA in a GTP-dependent manner and to deliver the aminoacyl-tRNA to the ribosome. Mutational analyses of eIF2␥ support a similar mode of aminoacyl-tRNA binding by eIF2␥ and EF-Tu, wherein the 3Ј (aminoacyl) end of the tRNA binds to domain II (3,5,6). Interestingly, the C-terminal end of eIF2␣ binds to an adjacent conserved surface on domain II (3, 7). The N terminus of the eIF2␤ subunit contains three lysine-rich segments (K-boxes) that are absent from the corresponding archaeal protein and do not appear to be important for binding to eIF2␥ (7,8). Moreover, the binding site for eIF2...

The x-ray structure of the ␥-subunit of the heterotrimeric translation initiation factor eIF2 has been determined to 2.4-Å resolution. eIF2 is a GTPase that delivers the initiator Met-tRNA to the P site on the small ribosomal subunit during a rate-limiting initiation step in translation. The structure of eIF2␥ closely resembles that of EF1A⅐GTP, consisting of an N-terminal G domain followed by two ␤-barrels arranged in a closed configuration with domain II packed against the G domain in the vicinity of the Switch regions. The G domain of eIF2␥ has an unusual zinc ribbon motif, not previously found in other GTPases. Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of eIF2␥ that bind the ␣-subunit and Met-tRNA i Met , respectively. These structural, biochemical, and genetic results provide new insights into eIF2 ternary complex assembly.Translation initiation involves assembly of a large protein-RNA complex at the initiation codon of a mRNA in three main steps: binding of initiator Met-tRNA and mRNA with associated factors to the small ribosomal subunit, pairing of the anticodon of Met-tRNA i Met with the AUG start codon, and joining of the two ribosomal subunits to form the 80 S initiation complex. In eukaryotes, this process requires at least twelve distinct translation initiation factors (eIFs) 1 and hydrolysis of two molecules of GTP (reviewed in Ref. 1). Translation initiation factor eIF2 is a heterotrimeric GTPase that delivers the Met-tRNA i Met to the small ribosomal subunit as part of a ternary complex with GTP. Pairing between the anticodon of the Met-tRNA i Met and the AUG start codon triggers hydrolysis of GTP by eIF2 and eIF2⅐GDP is released, leaving Met-tRNA i Met bound in the P site. eIF2⅐GDP cannot bind Met-tRNA i Met and is converted to eIF2⅐GTP by the heteropentameric exchange fac-eIF2 is a stable complex of three subunits (␣, ␤, and ␥), each essential for viability in yeast. Since their discovery, highly conserved eIF2 subunits have been identified in all eukaryotes (reviewed in Ref.2) and also archaebacteria (3). Biochemical and genetic analyses have demonstrated that the three subunits have distinct activities during translation initiation. The ␥-subunit binds GTP and Met-tRNA i Met (reviewed in Ref. 4). GTP hydrolysis by the eIF2 ternary complex bound to the 40 S ribosomal subunit is stimulated by eIF5, which interacts with eIF2␤ (5). The ␤-subunit also interacts with Met-tRNA i Met and has been reported to bind mRNA (6). Given the pivotal role eIF2 plays in eukaryotic translation initiation, it is not surprising that it represents an important target for regulation. In response to various environmental stressors (viral infection, amino acid starvation, heme deficiency, ER stress etc), the ␣-subunit is phosphorylated on Ser 51 , abolishing translation initiation by preventing eIF2B catalyzed GDP-GTP exchange.The ␥-subunit of eIF2 belongs to the superfamily of GTPbinding proteins and is most closely related to the translation elongation factor...

Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNAi Met and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNAi Met to the 40S ribosomal subunit, and previous studies identified Sui− mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNAi Met binding. Consistently, an eIF2γ-N135D GTP-binding domain mutation impairs Met-tRNAi Met binding and causes a Sui− phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNAi Met binding affinity and cell growth; however, only A208V suppresses the Sui− phenotype associated with the eIF2γ-N135D mutation. An eIF2γ-A219T mutation impairs Met-tRNAi Met binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui− phenotype associated with the eIF2γ-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui− phenotype of the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNAi Met on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNAi Met binding affinity.