The regulation of protein synthesis in mammalian cells under stress conditions as well as the regulation of GCN4-specific translation in the yeast Saccharomyces cerevisiae under amino acid starvation conditions is mediated by phosphorylation of the eukaryotic translation initiation factor (eIF)-2. Phosphorylation of serine-51 on the alpha subunit of eIF-2 by the mammalian PKR kinase inhibits general translation, while phosphorylation of serine-51 by the yeast GCN2 kinase is required to increase expression of GCN4. We have been studying how these protein kinases specifically recognize and phosphorylate eIF-2alpha on serine-51. Over 100 mutations throughout the eIF-2alpha protein have been identified that prevent induction of GCN4 expression when GCN2 is activated. Among the residues immediately flanking serine-51, the positions -1 and -2 appear most important for regulation. Examination of eIF-2alpha phosphorylation in vivo in strains carrying various mutant alleles reveals that some of the substitutions markedly affect the ability to phosphorylate serine-51. We have also been studying the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. We have established a system to suppress the toxicity of PKR expression in yeast by co- expressing K3L. Using this system both loss-of-function and increased activity K3L mutants have been isolated. Residues near the carboxyl- terminus of K3L, that are conserved in eIF-2alpha (residues 73-83), are critical for K3L activity. In addition, the corresponding residues in eIF-2alpha are required for proper regulation of GCN4 expression. Finally, we have begun characterization of a novel eIF-2alpha kinase inhibitor from the baculovirus Autographa californica.