Figure 4.

Tup1 repression occludes the holoenzyme and Tbp from the promoter. (A) Tup1 tightly regulates mating-specific gene expression. To determine the relative expression levels of a subset of mating type-specific genes we performed quantitative reverse transcription–PCR analysis on RNA isolated from the isogenic cells of opposite mating types. A constant amount of chromosomal DNA was used in the analysis of each gene to control for PCR efficiency of each primer pair. Actin gene expression was monitored to control for total RNA recovery. (B) Tup1 recruited to α2 repressed genes occludes the holoenzyme and Tbp from a-specific gene promoters. Chromosomal immunoprecipitation assays were performed on yeast cells of opposite mating type. Antibodies specific for PolII (anti-ctd), Gal11 (anti-Gal11) and Tbp (anti-Tbp) were used to immunoprecipitate in vivo formaldehyde cross-linked chromatin. Promoter DNA coupled to complexes containing PolII, Gal11, and Tbp were analyzed by quantitative PCR studies. Primer pairs encompassed the core promoter region of each gene. The histograms indicate DNA immunoprecipitated relative to the Actin promoter region for each gene. Plotted values correspond to the mean values obtained with three independent experiments. SEM = 15–25%.