The eukaryotic translation initiation factor eIF-2 plays a critical role in regulating the expression of the yeast transcriptional activator GCN4. Mutations in genes encoding the a and 1 subunits of eIF-2 alter translational efficiency at the GCN4 AUG codon and constitutively elevate GCN4 translation. Mutations in the yeast GCDII gene have been shown to confer a similar phenotype. The nucleotide sequence of the cloned GCDII gene predicts a 527-amino-acid polypeptide that is similar to the prokaryotic translation elongation factor EF-Tu. Relative to EF-Tu, the deduced GCD11 amino acid sequence contains a 90-amino-acid N-terminal extension and an internal cysteine-rich sequence that contains a potential metal-binding finger motif. We have identified the GCDII gene product as the 'y subunit of eIF-2 by the following criteria: (i) sequence identities with mammalian eIF-2'y peptides; (ii) increased eIF-2 activity in extracts prepared from cells cooverexpressing GCD11, eIF-2a, and eIF-21; and (iii) cross-reactivity of antibodies directed against the GCD11 protein with the 58-kDa polypeptide present in purified yeast eIF-2. The predicted GCD11 polypeptide contains all of the consensus elements known to be required for guanine nucleotide binding, suggesting that, in Saccharomyces cerevisiae, the 'y subunit of eIF-2 is responsible for GDP-GTP binding. tRNA, recognizes an mRNA (which is bound by an additional set of initiation factors) and attaches at or near its 5' capped end. The factor-bound 40S subunit then scans the mRNA linearly in a 5'-to-3' direction until it encounters an AUG translation initiation codon in a favorable context (41). Upon reaching the start codon, GTP is hydrolyzed, Met-tRNA, is donated to the ribosomal half-P site, and eIF-lA, eIF-3, and eIF-2-GDP are released from the 40S subunit in a reaction promoted by eIF-S. The eIF-2-GDP complex formed as a result of GTP hydrolysis must be converted to the GTP-bound form to allow eIF-2 to bind Met-tRNA, and participate in a subsequent round of initiation. The reaction in which GTP is exchanged for the GDP bound to eIF-2 is catalyzed by the guanine nucleotide exchange factor eIF-2B.In both amino acid-starved and unstarved cells, it appears that ribosomes initiate protein synthesis at the AUG codon of the 5'-proximal uORF (uORF1) on GCN4 mRNA, terminate three codons downstream at an in-frame termination codon, and dissociate from the mRNA. There is evidence, however, that about one-half of the 40S subunits that terminate at uORF1 remain associated with the GCN4 mRNA and resume scanning (1). (1,49). Amino acid starvation results in reduced reinitiation at uORFs 2, 3, and 4 and increased reinitiation at the GCN4 AUG codon, perhaps because of a decrease in the activity of one or more initiation factors (1).Genetic and molecular analyses have identified the initiation factor eIF-2 as a key regulator of GCN4 expression. In S. cerevisiae, as well as in mammals, eIF-2 is composed of three nonidentical subunits termed a