Equilibrium and kinetic models in gene regulation

Gene regulation in the cell is a complicated and tightly regulated process. The steps leading up to the creation of a gene product can be dominated by the equilibria or the kinetics of those steps. This thesis focuses on two regulators of gene expression: one that may be amenable to an equilibrium analysis, the other requiring a kinetic description. These regulators, nucleosomes and the TATA box binding protein (TBP) respectively, act during the initial steps leading up to the formation of a functional preinitiation complex (PIC).;We first investigated the early stages of competition between nucleosomes and transcription factors, in which two transcription factors cooperatively invade a nucleosome. This process is describable in terms of equilibria. The Widom laboratory has previously shown that one DNA binding factor can facilitate the binding of a second DNA binding factor if their binding sites are covered by the same half of one nucleosome, through a mechanism called collaborative competition. Here, we asked if this cooperativity occurs also when the binding sites are on opposite sides of the nucleosome. Using an in vitro quantitative restriction enzyme digestion assay, we show that collaborative competition does not extend across the middle (dyad axis) of the nucleosome. Our findings could help refine programs that aim to predict functional pairs of cooperative transcription factor binding sites in vivo.;We next explored the regulation of TBP, by the Mot1 ATPase. TBP is an essential component in PIC formation, and its lifetime on DNA is limited by Mot1. The ATPn-dependent removal of TBP from DNA by Mot1 requires a kinetic description. How Mot1 utilizes its ATP hydrolysis activity to drive TBP off DNA is not known. We discovered that upon Mot1 binding the DNA bound by TBP is unbent, even though TBP and DNA remain in close proximity. In addition, we discovered that Mot1's conserved Snf2/Swi2-family ATPase domain directly binds DNA, unifying the Mot1 mechanism with those of other Snf2/Swi2 family members. We integrated those findings to create a new model for Mot1 action. Together, these studies advance our understanding of both equilibrium and kinetic mechanisms in transcriptional regulation.