Monitoring the Gcn4 protein-mediated response in the yeast Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae the GCN4 gene encodes the transcriptional activator of the 'general control' system of amino acid bioynthesis, a network of at least 12 different biosynthetic pathways. We characterized the consequences of the general control response upon the signal 'amino acid starvation' induced by the histidine analogue 3-aminotriazole with respect to Gcn4p levels in more detail. Therefore, we established test systems to monitor the time course of different parameters, including GCN4 mRNA, Gcn4 protein, Gcn4p DNA binding activity, as well as Gcn4p transactivation ability. We observed a biphasic response of Gcn4p activity in the cell. At first, translation of GCN4 mRNA is induced within 20 min after switch to starvation conditions. However, an additional increase in GCN4 transcript steady state level was observed, leading to an additional second phase of GCN4 expression after 3-4 h of starvation. The DNA binding activity of Gcn4p, as well as the ability to activate transcription of target genes, correlate with the amount of Gcn4 protein in the cell, suggesting that under the tested conditions there is no additional regulation of DNA binding or transactivation ability of Gcn4p, respectively.