The Regulation and Analysis of Transcription Preinitiation Complex Dynamics
Wells, Melissa Nicole, Department of Biochemistry and Molecular Genetics, University of Virginia
Auble, David, Department of Biochemistry and Molecular Genetics, University of Virginia
Smith, Jeff, Department of Biochemistry and Molecular Genetics, University of Virginia
A central component of the transcription preinitiation complex is TATA binding protein (TBP). TBP has recently been shown to be highly dynamic in the nucleus. Our analysis of suggests that this dynamic behavior is critical for efficient RNA synthesis. When the dynamics of TBP are altered, sites of transcription initiation and termination are affected on a global scale. The efficiency of RNA synthesis is linked to the function of the Snf2/Swi2 ATPase Mot1. Mot1 removes TBP from DNA and ensures a mobile pool of TBP. In order to understand mechanistically how Mot1 promotes the efficiency of RNA synthesis, its connection with the histone chaperone FACT was investigated. The FACT complex regulates transcription initiation and elongation. Here, we show that Mot1 physically and genetically interacts with the FACT. They regulate the expression of many of the same genes. These results support a model in which Mot1 and FACT cooperate to remodel promoter nucleosomes, thereby facilitating transcriptional activation. In addition, a method has been developed to measure kinetics of site-specific chromatin binding genome-wide, in vivo. This method is based on a modified chromatin immunoprecipation assay and allows one to measure the on and off-rates of factors binding to DNA as well as their residence times and steady-state occupancies. Overall the work presented here extends our understanding of the role of chromatin binding dynamics in ensuring transcription efficiency and precision. In addition, the kinetic method presented here will likely be generally helpful for quantifying kinetic parameters of factors involved in transcription or other DNA metabolic processes. Publications iii The following papers correspond to the material presented in the indicated chapters.
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PHD (Doctor of Philosophy)
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