A carbon source-responsive promoter element necessary for activation of the isocitrate lyase gene ICL1 is common to genes of the gluconeogenic pathway in the yeast Saccharomyces cerevisiae.

The expression of yeast genes encoding gluconeogenic enzymes depends strictly on the carbon source available in the growth medium. We have characterized the control region of the isocitrate lyase gene ICL1, which is derepressed more than 200-fold after transfer of cells from fermentative to nonfermentative growth conditions. Deletion analysis of the ICL1 promoter led to the identification of an upstream activating sequence element, UASICL1 (5' CATTCATCCG 3'), necessary and sufficient for conferring carbon source-dependent regulation on a heterologous reporter gene. Similar sequence motifs were also found in the upstream regions of coregulated genes involved in gluconeogenesis. This carbon source-responsive element (CSRE) interacts with a protein factor, designated Ang1 (activator of nonfermentative growth), detectable only in extracts derived from derepressed cells. Gene activation mediated by the CSRE requires the positively acting derepression genes CAT1 (= SNF1 and CCR1) and CAT3 (= SNF4). In the respective mutants, Ang1-CSRE interaction was no longer observed under repressing or derepressing conditions. Since binding of Ang1 factor to the CSRE could be competed for by an upstream sequence derived from the fructose-1,6-bisphosphatase gene FBP1, we propose that the CSRE functions as a UAS element common to genes of the gluconeogenic pathway.     

Identification and characterization of regulatory elements in the phosphoenolpyruvate carboxykinase gene PCK1 of Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase is a key enzyme in gluconeogenesis. The expression of the PCK1 gene in Saccharomyces cerevisiae is strictly regulated and dependent on the carbon source provided. Two upstream activation sites (UAS1_PCK1 and UAS2_PCK1) and one upstream repression site (URS_PCK1) were localized by detailed deletion analysis. The efficacy of these three promoter elements when separated from each other was confirmed by investigations using heterologous promoter test plasmids. Activation mediated by UAS1_PCK1 or UAS2_PCK1 did not occur in the presence of glucose, indicating that these elements are essential for glucose derepression. The repressing effect caused by URS_PCK1 was much stronger in glucose-grown cells than in ethanol-grown cells.     

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter

Summary The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides –676 to –381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides –513 to –501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5 deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 by sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions –486 to –480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRFl UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the –676 to –381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.     

Identification and characterization of regulatory elements in the phosphoenolpyruvate carboxykinase gene PCK1 of Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase is a key enzyme in gluconeogenesis. The expression of the PCK1 gene in Saccharomyces cerevisiae is strictly regulated and dependent on the carbon source provided. Two upstream activation sites (UAS1_PCK1 and UAS2_PCK1) and one upstream repression site (URS_PCK1) were localized by detailed deletion analysis. The efficacy of these three promoter elements when separated from each other was confirmed by investigations using heterologous promoter test plasmids. Activation mediated by UAS1_PCK1 or UAS2_PCK1 did not occur in the presence of glucose, indicating that these elements are essential for glucose derepression. The repressing effect caused by URS_PCK1 was much stronger in glucose-grown cells than in ethanol-grown cells.