Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2␣ (eIF2␣) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2␣, including those at residues flanking Ser51 and around 20 Å away in the conserved motif K 79 GYID 83 . Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2␣. Thus, two structurally distinct effectors of eIF2 function, eIF2␣ kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2␣.

The family of eukaryotic initiation factor 2␣ (eIF2␣) protein kinases plays an important role in regulating cellular protein synthesis under stress conditions. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate Ser-51 on the ␣ subunit of the translation initiation factor eIF2. By using an in vivo assay in yeast, the substrate specificity of these three eIF2␣ kinases was examined by substituting Ser-51 in eIF2␣ with Thr or Tyr. In yeast, phosphorylation of eIF2 inhibits general translation but derepresses translation of the GCN4 mRNA. All three kinases phosphorylated Thr in place of Ser-51 and were able to regulate general and GCN4-specific translation. In addition, both PKR and HRI were found to phosphorylate eIF2␣-S51Y and stimulate GCN4 expression. Isoelectric focusing analysis of eIF2␣ followed by detection using anti-eIF2␣ and anti-phosphotyrosine-specific antibodies demonstrated that PKR and HRI phosphorylated eIF2␣-S51Y on Tyr in vivo. These results provide new insights into the substrate recognition properties of the eIF2␣ kinases, and they are intriguing considering the potential for alternate substrates for PKR in cellular signaling and growth control pathways.The human interferon-induced double-stranded RNA-activated protein kinase PKR, which functions in the cellular antiviral defense mechanism, is a member of a family of structurally related Ser/Thr kinases that specifically phosphorylate Ser-51 on the ␣ subunit of the translation initiation factor eIF2 1 (1, 2). The binding of double-stranded RNA, thought to be generated during viral infections, is proposed to alter the conformation of PKR and activate the kinase to autophosphorylate (1, 2). The active, phosphorylated form of PKR can then phosphorylate eIF2␣ on Ser-51 and convert eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B, resulting in the inhibition of translation initiation (1, 2). The other members of the eIF2␣ kinase family are the mammalian heme-regulated inhibitor of translation (HRI) that is activated by heme deprivation, the apparently ubiquitous kinase GCN2, first identified in yeast but also found in flies and mammals, which is activated under conditions of amino acid or purine nucleotide deprivation (1-4), and the newly identified mammalian kinase PERK or PEK, a transmembrane kinase located in the endoplasmic reticulum that is activated under conditions of endoplasmic reticulum stress (5, 6). In the yeast Saccharomyces cerevisiae, low level phosphorylation of eIF2␣ by GCN2 alters the pattern of translation reinitiation on the GCN4 mRNA and induces GCN4 expression (2). Increased synthesis of GCN4, a transcriptional activator of amino acid biosynthetic genes, enables cells to withstand amino acid starvation conditions. The mammalian eIF2␣ kinases PKR and HRI can substitute for GCN2 in yeast to phosphorylate eIF2␣ and stimulate GCN4 translation (7). In addition, high level phosphorylation of eIF2␣ in yeast by mutationally hyperactivated alleles of GCN2 or by overexpression of PK...

TranscriptionaUly active regions of the Haloferax vokcanii genome were mapped by hybridization of radiolabeled cDNA to Southern blots of our minimal set of overlapping cosmid clones covering 96% of the 4.1-Mbp genome. Transcription during exponential growth occurred in nearly every region of the 2,920-kbp chromosome. Large parts of the 690-and the 86-kbp plasmids were transcribed, but the 440-kbp plasmid showed little expression. Transcription after a 40-min heat shock at 65°C was generally reduced, apart from a smal set of strongly expressed loci all situated on the chromosome.A high-resolution physical map of the 4.1-Mbp genome of the archaebacterium Haloferax volcanii DS2 has been recently completed (3). The genome is partitioned into five circular replicons: a chromosome of 2,920 kbp and four plasmids of 690, 440, 86, and 6.4 kbp. The detailed study of halobacterial genome structure is useful in that it provides an archaebacterial data base for comparative genome analysis, and it addresses the issue of the dynamics and evolution of a genome rich in insertion elements (4,8,15,16) and plasmids.We and our collaborators are interested in mapping genes to the physical framework of overlapping cosmid clones which currently cover 96% of the H. volcanii genome. The following three approaches have been used: (i) hybridization of Southern blots of digested cosmid clones by using bulk probes (tRNA or insertion elements) has allowed the mapping of families of genes (4), (ii) individual cloned halobacterial genes have been mapped by homologous or heterologous hybridization (3), and (iii) complementation of auxotrophic mutants with cosmid clone DNA has mapped 140 mutations to 35 loci (4,5,10).With the goal of further understanding the genome of H. volcanii, we designed an experiment to find those loci most transcriptionally active under different physiological conditions by synthesizing radiolabeled cDNA and hybridizing it to Southern blots of digests of our minimal set of overlapping cosmid clones. This report describes the transcriptional organization of the genome of a culture in exponential growth (in chemically defined medium [CDM] and in complex medium) and compares it with that of a culture experiencing heat shock. MATERIALS AND METHODSGrowth media and conditions. CDM was a modification of that described by Kauri et al. (9) in which glycerol and succinate were replaced with 30 mM trisodium citrate. Rich medium was as described in reference 6. Cultures (50 ml) were grown at 45°C with shaking in 250-ml Erlenmeyer flasks. Cells were harvested during exponential growth (A540, 1.6) in rich medium, during exponential growth (A540, * Corresponding author.1.0) in CDM, or after exponential growth (A540, 1.0) in CDM followed by a 40-min heat shock at 65°C with occasional agitation.RNA extraction. Cells were quick-chilled to -15°C by immersing and swirling the flask for 2 min in a salted ice-water bath before the culture was centrifuged at -10 to -15°C for 10 min at 6,000 x g. The cell pellet was suspended in 0.25 ml of col...