Contribution of the Saccharomyces cerevisiae transcriptional regulator Leu3p to physiology and gene expression in nitrogen- and carbon-limited chemostat cultures

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Abstract
Transcriptional regulation of branched-chain amino-acid metabolism in Saccharomyces cerevisiae involves two key regulator proteins, Leu3p and Gcn4p. Leu3p is a pathway-specific regulator, known to regulate six genes involved in branched-chain amino-acid metabolism and one gene in nitrogen assimilation. Gcn4p is a global regulator, involved in the general response to amino-acid and purine starvation. To investigate the contribution of Leu3p in regulation of gene expression, a leu3Δ strain was compared to an isogenic reference strain using DNA-microarray analysis. This comparison was performed for both glucose-grown/ammonium-limited and ethanol-limited/ammonium-excess chemostat cultures. In ethanol-limited cultures, absence of Leu3p led to reduced transcript levels of six of the seven established Leu3p target genes, but did not affect key physiological parameters. In ammonium-limited cultures, absence of Leu3p caused a drastic decrease in storage carbohydrate content. mRNA levels of genes involved in storage carbohydrate metabolism were also found reduced. Under N-limited conditions, the leu3Δ genotype elicited an amino-acid starvation response, leading to increased transcript levels of many amino-acid biosynthesis genes. By combining the transcriptome data with data from earlier studies that measured DNA binding of Leu3p both in vitro and in vivo, BAT1, GAT1 and OAC1 were identified as additional Leu3p-regulated genes. This study demonstrates that unravelling of transcriptional regulation networks should preferably include several cultivation conditions and requires a combination of experimental approaches.