TATA binding protein (TBP) plays a central role in transcription complex assembly and is regulated by a variety of transcription factors, including Mot1. Mot1 is an essential protein in Saccharomyces cerevisiae that exerts both negative and positive effects on transcription via interactions with TBP. It contains two conserved regions important for TBP interactions, another conserved region that hydrolyzes ATP to remove TBP from DNA, and a fourth conserved region with unknown function. Whether these regions contribute equally to transcriptional regulation genome-wide is unknown. Here, we employ a transient-replacement assay using deletion derivatives in the conserved regions of Mot1 to investigate their contributions to gene regulation throughout the S. cerevisiae genome. These four regions of Mot1 are essential for growth and are generally required for all Mot1-regulated genes. Loss of the ATPase region, but not other conserved regions, caused TBP to redistribute away from a subset of Mot1-inhibited genes, leading to decreased expression of those genes. A corresponding increase in TBP occupancy and expression occurred at another set of genes that are normally Mot1 independent. The data suggest that Mot1 uses ATP hydrolysis to redistribute accessible TBP away from intrinsically preferred sites to other sites of intrinsically low preference.