My project focuses on the mechanism of a yeast protein known as Mot1p, a molecular machine that utilizes the hydrolysis of ATP to disrupt TBP-DNA complexes. We are interested in Mot1p for two reasons. First, Mot1p is an essential regulator of transcription. It has the remarkable property of increasing the off-rate of TBP from DNA. TBP is essential for the formation of the pre-initiation complex (PIC), yet TBP-DNA complexes do not associate and dissociate on the timescale necessary to be a dynamic regulator of transcription. Therefore, by increasing the off rate of TBP from DNA, Mot1p directly controls the lifetime of the ‘on’ state of a gene. Second, Mot1p is a member of the SNF2/SWI2 ATPase family for which no mechanisms are currently known. Mot1p is an especially useful system to study the mechanism of this ATPase superfamily because of the relative simplicity of the system: the only requirements are TBP, DNA and Mot1p. I propose to use biophysical techniques including fluorescence anisotropy, surface plasmon resonance, and steady state, stopped flow, single molecule and FCS FRET to address unanswered questions regarding various functional domains of Mot1p and to test current models of Mot1p catalyzed disruption of TBP-DNA complexes.
