Abstract
Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.
Highlights
In bakers’ yeast, Saccharomyces cerevisiae, starvation for a single amino acid induces the transcription of more than 50 genes encoding enzymes involved in several amino acid biosynthetic pathways, amino acid tRNA synthetases [1, 2], or enzymes of purine biosynthesis [3]
The ⌺1278b background is ideal for measuring the effects of nitrogen starvation, because ⌺1278b strains are highly responsive to changes in the ammonium concentrations in the environment
The GCRE6::lacZ gene was integrated into the URA3 locus of wild-type control and gcn4⌬ mutant strains of both genetic backgrounds. -Galactosidase activities of resulting strains were determined under three different growth conditions as follows: mid-log phase, starvation for amino acids by addition of the histidine analogue 3AT, and starvation for nitrogen by 1000-fold reduction of the ammonium sulfate concentration (Fig. 1)
Summary
MATa/␣ ura3–52/ura3–52ϻGCRE6ϻlacZϻURA3 his3ϻhisG/HIS3 trp1ϻhisG/trp1ϻhisG MATa/␣ ura3–52/ura3–52ϻGCRE6ϻlacZϻURA3 leu2ϻhisG/leu2ϻhisG his3ϻhisG/HIS3. LEU2/leu leu112 gcn4⌬/gcn101 (S288c-background) MATa/␣ ura3–52/ura3–52ϻGCRE6ϻlacZϻURA3 his3ϻhisG/HIS3 trp1ϻhisG/trp1ϻhisG aro3⌬ϻkanR/aro3⌬ϻkanR MATa/␣ ura3–52/ura3–52ϻGCRE6ϻlacZϻURA3 his3ϻhisG/HIS3 trp1ϻhisG/trp1ϻhisG aro3⌬ϻkanR/aro3⌬ϻkanR ARO4m/ARO4m MATa/␣ ura3–52/ura3–52ϻGCRE6ϻlacZϻURA3 his3ϻhisG/HIS3 trp1ϻhisG/trp1ϻhisG ure2⌬ϻkanR/ure2⌬ϻkanR MATa/␣ ura3–52/ura3–52ϻGCRE6ϻlacZϻURA3 his3ϻhisG/HIS3 trp1ϻhisG/trp1ϻhisG gln3⌬ϻkanR/gln3⌬ϻkanR MATa ura leu leu112 trp1-⌬63 gcn3⌬ϻLEU2 (S288c-background) MATa ura (S288c-background). Expression of a synthetic GCRE6::lacZ reporter gene and natural amino acid biosynthetic genes such as ARO4 is suppressed when cells are starved for nitrogen, whereas starvation for glucose has only minor effects on Gcn4p-controlled gene expression. We find GCRE-mediated repression the mRNA level of GCN4 strongly increases, suggesting that nitrogen starvation suppresses translation of GCN4. The four intact uORFs in the GCN4 mRNA leader sequence are necessary components for the repression mechanism, whereas eIF-2␣ phosphorylation, the ammonium sensing system that includes Mep2p or Gpa2p, and the proteins Ure2p or Gln3p are not required
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