The broad, long-term objectives of the proposed project are to elucidate mechanisms responsible for assembly and activity of RNA polymerase II (Pol II) preinitiation complexes (PICs) at promoters. The TATA- binding protein (TBP), a central component of the PIC, can bind to DNA in vitro with exceptional stability. However, essentially all of the TBP in yeast cells is highly dynamic, with residence times on chromatin of just a few seconds. Prevailing models derived from in vitro observations posit that a stable promoter-bound complex facilitates transcription reinitiation and thereby perpetuates the activated state. In contrast, we find that dynamic turnover is critical for global transcriptional control in vivo. TBP's dynamic behavior is a consequence of the activity of Mot1, an essential, conserved, Snf2/Swi2-related ATPase that uses the energy of ATP hydrolysis to dissociate TBP from DNA. The goals of this proposal are to use Mot1 as a model to better understand how members of this critical class of enzymes use ATP hydrolysis to disrupt protein-DNA interactions, and to understand why Mot1-mediated dynamics are essential for transcription in vivo. In the first Aim, the Mot1 biochemical system and recent structural data will be exploited to test a specific model for the Mot1 catalytic mechanism. Emerging evidence indicates that cell-to-cell gene expression variation (""""""""noise"""""""") impacts a wide range of cellular responses to stress and developmental cues, but mechanisms regulating noise are not well understood. Preliminary results show that Mot1 has a previously unappreciated role in suppressing transcriptional noise across a cell population.
In Aim 2, we will define the scope and function of Mot1 and other related global regulators in establishing noise levels, and correlate this variation with PIC dynamics.
In Aim 3, we will define a newly discovered functional relationship between Mot1 and FACT, an essential regulator of chromatin structure and dynamics. In addition to determining how Mot1 and FACT work together in vivo, new functions of Mot1 in chromatin structure regulation will be addressed, including the unexpected ability of Mot1 to bind to nucleosomes and to regulate the interaction of FACT with nucleosomes. Precise transcriptional control is essential for normal cell growth and development, and numerous transcriptional defects have been linked to human diseases including cancer. Defects in human Snf2/Swi2- related protein complexes are known contributors to certain cancers, Cockayne's Syndrome, alpha- thalassemia, and the most common form of X-linked mental retardation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics A Study Section (MGA)
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Sledjeski, Darren D
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University of Virginia
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Viswanathan, Ramya; True, Jason D; Auble, David T (2016) Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme. J Biol Chem 291:15714-26
True, Jason D; Muldoon, Joseph J; Carver, Melissa N et al. (2016) The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization. J Biol Chem 291:15307-19
Hoffman, Elizabeth A; Frey, Brian L; Smith, Lloyd M et al. (2015) Formaldehyde crosslinking: a tool for the study of chromatin complexes. J Biol Chem 290:26404-11
Butryn, Agata; Schuller, Jan M; Stoehr, Gabriele et al. (2015) Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1. Elife 4:
Viswanathan, Ramya; Hoffman, Elizabeth A; Shetty, Savera J et al. (2014) Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method. Methods 70:97-107
Poorey, Kunal; Viswanathan, Ramya; Carver, Melissa N et al. (2013) Measuring chromatin interaction dynamics on the second time scale at single-copy genes. Science 342:369-72
Moyle-Heyrman, Georgette; Viswanathan, Ramya; Widom, Jonathan et al. (2012) Two-step mechanism for modifier of transcription 1 (Mot1) enzyme-catalyzed displacement of TATA-binding protein (TBP) from DNA. J Biol Chem 287:9002-12
Wollmann, Petra; Cui, Sheng; Viswanathan, Ramya et al. (2011) Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP. Nature 475:403-7
Hyland, Edel M; Molina, Henrik; Poorey, Kunal et al. (2011) An evolutionarily 'young' lysine residue in histone H3 attenuates transcriptional output in Saccharomyces cerevisiae. Genes Dev 25:1306-19
Viswanathan, Ramya; Auble, David T (2011) One small step for Mot1; one giant leap for other Swi2/Snf2 enzymes? Biochim Biophys Acta 1809:488-96

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