cpc-3, the Neurospora crassa homologue of yeast GCN2, encodes a polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains required for general amino acid control.
Based on characteristic amino acid sequences of kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha kinases), degenerate oligonucleotide primers were constructed and used to polymerase chain reaction-amplify from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase. With this sequence an open reading frame was identified encoding a predicted polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains. The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional activator of amino acid biosynthetic genes encoded by GCN4. Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived cells. The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1, encoding the GCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N. crassa, but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation. These findings suggest that cpc-3 is the functional homologue of GCN2, being required for increased translation of cpc-1 mRNA in amino acid-starved cells.
Sattlegger
E
E
Institute of Applied Genetics, University of Hannover, Herrenhäuser Strasse 2, D-30419 Hannover, Germany. esattlegger@aghmac1.nichd.nih.gov
Hinnebusch
A G
AG
Barthelmess
I B
IB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis*; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D006643:Histidine-tRNA Ligase / Q000737:chemistry*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology*; D009954:Ornithine Carbamoyltransferase / Q000235:genetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation / Q000235:genetics
D000596:Amino Acids; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D005656:Fungal Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D009954:Ornithine Carbamoyltransferase; D011494:Protein Kinases; C114066:cpc-3 protein, Neurospora crassa; D006643:Histidine-tRNA Ligase