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TSF3 encodes one of six (TSF1 to TSF6) recently identified global negative regulators of transcription in Saccharomyces cerevisiae. Mutant tsf3 strains exhibit defects in transcriptional silencing of the GAL1 promoter, allow expression from upstream activation sequence-less promoters, and exhibit pleiotropic defects in cell growth and development. Here we show that TSF3 is involved in transcriptional silencing mediated by the alpha 2 repressor and demonstrate that specific systems of transcriptional silencing may depend on the more global role of TSF3. Cloning and sequencing of TSF3 allowed us to predict a 974-amino-acid gene product identical to SIN4, a negative regulator of transcription of the HO (homothallism) mating type switching endonuclease. TSF3 disruptions are not lethal but result in phenotypes similar to those of the originally isolated alleles. Our results, together with those of Y. W. Jiang and D. J. Stillman (Mol. Cell. Biol. 12:4503-4514, 1992), suggest that TSF3 (SIN4) affects the function of the basal transcription apparatus, and this effect in turn alters the manner in which the latter responds to upstream regulatory proteins.