Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.     

Evidence that the GCN2 protein kinase regulates reinitiation by yeast ribosomes.

The yeast gene GCN4 produces an mRNA that has a long 5' 'untranslated' region containing four small open reading frames (ORFs) preceding the protein coding frame. This configuration suppresses the rate by which GCN4 protein is synthesized. However, translational derepression of the GCN4 mRNA occurs when yeast cells are grown under conditions of amino acid limitation. Such translational derepression requires the GCN2 protein kinase and the presence of the 5' most proximal ORF. In this study we show that a functional coupling between the translation of the first ORF and the amount of the GCN2 protein is responsible for the translational derepression of the GCN4 mRNA. Our evidence suggests that this coupling involves an increase in the ability of 40S ribosomal subunits that have translated the first frame to resume scanning and reinitiate translation at a downstream AUG independently of the base sequence in the intervening region.