Arginine restriction induced by delta-N-(phosphonacetyl)-L-ornithine signals increased expression of HIS3, TRP5, CPA1, and CPA2 in Saccharomyces cerevisiae.
delta-N-(Phosphonacetyl)-L-ornithine (PALO), a transition state analog inhibitor of ornithine transcarbamylase, induced arginine limitation in vivo in Saccharomyces cerevisiae. Arginine restriction caused increased expression of HIS3 and TRP5, measured by the beta-galactosidase activity in strains carrying chromosomally integrated fusions of the promoter regions of each gene with the lacZ gene of Escherichia coli. The increase in beta-galactosidase activity induced by PALO was reversed by the addition of arginine and was dependent on GCN4 protein. These results indicate that PALO, like 3-amino-1,2,4-triazole DL-5-methyltryptophan, can be used to study the effect of limitation of a single amino acid, arginine, on the expression of genes under the general amino acid control regulatory system. Arginine deprivation imposed by PALO also caused increased expression of CPA1 and CPA2, coding respectively for the small and large subunits of arginine-specific carbamyl-phosphate synthetase. The observed increase was GCN4 dependent and was genetically separable from arginine-specific repression of CPA1 mRNA translation. The 5'-flanking regions of CPA1 (reported previously) and CPA2 determined in this study each contained at least two copies of the sequence TGACTC, shown to bind GCN4 protein. The beta-galactosidase activities expressed from CPA1- and CPA2-lacZ fusions integrated into the nuclear DNA of gcn4 mutant strains were five to six times less than in the wild type, when both strains were grown under depressed conditions. The gcn4 mutation reduced basal expression of both CPA1 and CPA2. The addition of arginine to strains containing the CPA1-lacZ fusion further reduced beta-galactosidase activity of the gcn4 mutant, indicating independent regulation of the CPA1 gene by the general amino acid control and by arginine-specific repression. In strains overproducing GCN4 protein, the translational control completely overrode transcriptional activation of CPA1 by general amino acid control.
Kinney
D M
DM
Department of Molecular Genetics, Public Health Research Institute, New York, New York 10016.
Lusty
C J
CJ
D000596:Amino Acids / Q000502:physiology; D001120:Arginine / Q000378:metabolism* / Q000502:physiology; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007700:Kinetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D009154:Mutation; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
D000596:Amino Acids; C020464:N(delta)-(phosphonoacetyl)-L-ornithine; D001120:Arginine; D009952:Ornithine; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)