title: Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.
abstract: The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.
authors.affiliation: Section on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: M
authors.initials: M
authors.lastname: Ramirez
authors.forename: R C
authors.initials: RC
authors.lastname: Wek
authors.forename: C R
authors.initials: CR
authors.lastname: Vazquez de Aldana
authors.forename: B M
authors.initials: BM
authors.lastname: Jackson
authors.forename: B
authors.initials: B
authors.lastname: Freeman
authors.forename: A G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal; D006643:Histidine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010445:Peptide Elongation Factors; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins; C501389:GCN1 protein, S cerevisiae; C501390:GCN3 protein, S cerevisiae; D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D006643:Histidine-tRNA Ligase