Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA iMet to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2⅐GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The ␣-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2␤␥) completely devoid of the ␣-subunit to further study the role of eIF2␣ in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2␣ structural gene (SUI2). Removal of the ␣-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA i Met ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2␤␥ and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the ␣-subunit increased K m for eIF2␤␥⅐GDP by an order of magnitude, with a smaller increase in V max . These data indicate that eIF2␣ is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange. We suggest that this function contributes to the ability of the ␣-subunit to control the rate of nucleotide exchange through reversible phosphorylation. Eukaryotic translation initiation factor eIF21 is a heterotrimeric GTP-binding protein that in yeast is encoded by the essential genes SUI2, SUI3, and GCD11. eIF2 binds charged initiator tRNA (Met-tRNA i Met ) in a GTP-dependent manner to form a stable ternary complex that interacts with the small ribosomal subunit and additional initiation factors (including eIF3 and eIF1A) to form a 43 S initiation complex (reviewed in Refs. 1 and 2-5). This complex binds at or near the 5Ј-end of capped mRNAs and "scans" the mRNA in a 5Ј to 3Ј direction until an AUG start codon in proper context is encountered. Genetic analyses in yeast have demonstrated a function for eIF2 in the selection of the translational start site (6, 7). Recognition of the start codon is accompanied by eIF5-mediated hydrolysis of eIF2-bound GTP, which facilitates release of an inactive eIF2⅐GDP binary complex as well as several other initiation factors. The 40 S/mRNA/Met-tRNA iMet complex interacts with a 60 S ribosomal subunit to form an 80 S initiation complex that can then enter the elongation phase of protein synthesis. Conversion of the inactive eIF2⅐GDP binary complex to eIF2⅐GTP, which is capable of rebinding initiator tRNA, is catalyzed by the nucleotide exchange factor eIF2B.To execute its functions in translation initiation, eIF2 must interact with multiple ligands as well as other components of the translational apparatus. The former include guanine nucleotides and initi...

Eukaryotic translation initiation factor 2 (eIF2) is a G protein heterotrimer required for GTP-dependent delivery of initiator tRNA to the ribosome. eIF2B, the nucleotide exchange factor for eIF2, is a heteropentamer that, in yeast, is encoded by four essential genes and one nonessential gene. We found that increased levels of wild-type eIF2, in the presence of sufficient levels of initiator tRNA, overcome the requirement for eIF2B in vivo. Consistent with bypassing eIF2B, these conditions also suppress the lethal effect of overexpressing the mammalian tumor suppressor PKR, an eIF2α kinase. The effects described are further enhanced in the presence of a mutation in the G protein (γ) subunit of eIF2, gcd11-K250R, which mimics the function of eIF2B in vitro. Interestingly, the same conditions that bypass eIF2B also overcome the requirement for the normally essential eIF2α structural gene (SUI2). Our results suggest that the eIF2βγ complex is capable of carrying out the essential function(s) of eIF2 in the absence of eIF2α and eIF2B and are consistent with the idea that the latter function primarily to regulate the level of eIF2·GTP·Met-tRNAiMet ternary complexes in vivo.