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Our characterization of the Saccharomyces cerevisiae TATA box-binding protein (TBP) has led to the identification of nine specific yeast TBP-associated factors (TAFs) ranging in size from 170 to 25 kDa. The amino acid sequence derived from a purified TAF with an apparent M(r) of 170,000 indicates that yeast Taf170 is encoded by the essential yeast gene MOT1. We describe in this report a series of experiments that demonstrate that the protein encoded by MOT1 is a bona fide yeast TAF and that Taf170 forms a separate complex with TBP distinct from the RNA polymerase II-specific multisubunit transcription factor IID TBP-TAF complex. The significance of this unique TBP-Taf170 complex regarding transcriptional regulation is discussed.