Identification of cis-acting elements in the SUC2 promoter of Saccharomyces cerevisiae required for activation of transcription

We analyzed the effects of site-directed mutations in the SUC2 promoter of Saccharomyces cerevisiae. Analyses were performed in wild-type as well as mig1 and tup1 mutant strains after the promoter mutants were reintroduced into the native SUC2 locus on the left arm of chromosome IX. Mutation of the two GC boxes revealed that these elements play two distinct roles: they are, as expected, required for Mig1-mediated repression but they are also necessary for activation of the SUC2 promoter in response to glucose limitation. The individual GC boxes are functionally redundant with regard to Mig1-mediated repression, however, only the upstream GC box is essential for high level expression of SUC2. Microccocal nuclease sensitivity of the SUC2 promoter in derepressed cells was reduced in the GC box mutant promoters, particularly in the vicinity of the TATA box. The difference in nuclease sensitivity between wild-type and GC box mutant promoters was not evident in tup1- cells. The formation of nuclease-resistant chromatin does not require the GC boxes, indicating that other cis-acting elements can serve to recruit the Ssn6-Tup1 co-repressor complex to the SUC2 promoter.     

The anatomy of a hypoxic operator in Saccharomyces cerevisiae

Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.