Transcriptional Regulation by Mot1 and Spt16

True, Jason, Biochemistry and Molecular Genetics - Graduate School of Arts and Sciences, University of Virginia
Auble, David, Department of Biochemistry and Molecular Genetics, University of Virginia
Smith, Jeffrey, Department of Biochemistry and Molecular Genetics, University of Virginia
Grant, Patrick, Department of Biochemistry and Molecular Genetics, University of Virginia
Pemberton, Lucy, Department of Microbiology, University of Virginia

The Saccharomyces cerevisiae ATPase Mot1 regulates transcription by impacting the distribution and activity of TATA-binding protein (TBP). In vitro, Mot1 forms a complex with TBP and DNA, and uses ATP hydrolysis to dissociate TBP from DNA. To gain insight into the Mot1 mechanism, we employed a DNA tethered cleavage approach to map regions of Mot1 in proximity to DNA under different conditions. We present evidence for two conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence. We also show using purified complexes that Mot1 dissociation of TBP-DNA is inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. In addition, Mot1 and the Spt16 component of the FACT histone chaperone complex, have been shown to physically interact. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in S. cerevisiae and physically and genetically interact. Mot1 controlled TBP levels at co-regulated promoters, whereas Spt16 did not. Both Mot1 and Spt16 contribute to TFIIB localization, indicating a convergence on preinitiation complex formation. Globally, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization. Interestingly, we found that Mot1 has a role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5’ ends of genes. Spt16 has a broad role in regulating chromatin organization, including those nucleosomes affected by Mot1. These results suggest that Spt16 is required for Mot1 localization to the promoter by establishing a permissive chromatin environment for Mot1. Overall these results suggest that the overlap in Mot1 and Spt16 function arises from a combination of their unique and shared functions in transcription complex assembly.

PHD (Doctor of Philosophy)
Transcription, Mot1, Spt16
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