The Role of RAP1 in the Regulation of the MATα Locus

Abstract

The RAP1 gene of Saccharomyces cerevisiae encodes an abundant DNA-binding protein, also known as GRF1, TBA, or TUF, that binds to many sites in the yeast genome in vitro. These sites define a consensus sequence, 5′-^AA_GCCCCANNCA^TT_CC-3′ and deletion analyses of genes that contain this sequence have implicated the involvement of RAP1 in numerous cellular processes, including gene activation and repression. The MATα locus, required for determination of the a cell type in yeast cells, contains a RAP1 binding site; this site coincides with the MATα upstream activating sequence (UAS) and is necessary for expression of the two genes encoded by the MATα locus, MATα and MATα2. We show that the MATα UAS is sufficient to activate transcription from a promoterless gene fusion of the yeast CYC1 upstream region and the lacZ gene. Constructs containing only the MATα UAS generated elevated levels of β-galactosidase activity which were indistinguishable from those of constructs containing the entire MATα intergenic region. Further, the MATα UAS has an intrinsic polarity of transcriptional activation; transcription of CYC1-lacZ was six- to sevenfold higher when the UAS was oriented in the direction normally associated with MATα2 transcription. Point mutations in the MATα UAS that reduce MATα expression three- to fivefold resulted in a bi-mating phenotype, while a mutation that reduced MATα expression still further resulted in an a-mating phenotype. We isolated plasmids from a high-copy-number yeast library that suppressed the bi-mating defect of point mutations in the MATα UAS, and the most effective dosage suppressor contained the gene encoding RAP1. A temperature-sensitive rap1 mutant bi-mates at the semipermissive temperature. Double mutants at rap1 and MATα mate exclusively as a cells, at all temperatures, and do not express detectable levels of MATα RNA. These data provide evidence that the RAP1 gene product functions at the MATα UAS in vivo.