Cooperative action of NC2 and Mot1p to regulate TATA-binding protein function across the genome

Promoter recognition by TATA-binding protein (TBP) is an essential step in the initiation of RNA polymerase II (pol II) mediated transcription. Genetic and biochemical studies in yeast have shown that Motlp and NC2 play important roles in inhibiting TBP activity. To understand how TBP activity is regulated in a genome-wide manner, we profiled the binding of TBP, NC2, Motlp, TFIID, SAGA, and pol II across the yeast genome using chromatin immunoprecipitation (ChIP)-chip for cells in exponential growth and during reprogramming of transcription. We find that TBP, NC2, and Motlp colocalize at transcriptionally active pol II core promoters. Relative binding of NC2α and Motlp is higher at TATA promoters, whereas NCβ has a preference for TATA-less promoters. In line with the ChIP-chip data, we isolated a stable TBP-NC2-Mot1p-DNA complex from chromatin extracts. ATP hydrolysis releases NC2 and DNA from the Mot1p-TBP complex. In vivo experiments indicate that promoter dissociation of TBP and NC2 is highly dynamic, which is dependent on Mot1p function. Based on these results, we propose that NC2 and Motlp cooperate to dynamically restrict TBP activity on transcribed promoters.