Hewett University Professor
Hamilton Hall 737
402.472.3316
lparkhur@unlserve.unl.edu

Parkhurst Research Group
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Current Research
Our lab is working to understand fundamental processes in the initiation of eukaryotic transcription. Our goal is to understand the structural and energetic features that control the recognition of DNA promoter sequences by TBP (TATA Binding Protein) and the further assembly of the multi-protein transcription initiation complex. We use numerous approaches that include microcalorimetry, steady-state and time-resolved fluorescence and anisotropy in the psec-nsec time regime, stopped-flow rapid reaction kinetics, and molecular dynamic studies of protein-DNA interactions using the university supercomputer.

Using these various techniques, we have shown that the binding of DNA by TBP is a multi-step process, depicted below, in which DNA is strongly bent (by about 80o) in the very first detectable complex, one that we have proposed is the crucial intermediate involved in building the pre-initiation complex. We have used time-resolved fluorescence energy transfer (FRET) to show that the bending of the DNA is sequence dependent and correlates with the activity of the promoter in transcription.

Using pulsed laser techniques, we have developed time-resolved acceptor FRET to measure distances in protein complexes in solution that exceed 100. At right is the ternary complex of TBP-DNA-TFIIA, with the distance between arrows at sites "A" and "B" corresponding to 115, a distance we have measured between dyes fluorescein (F) and xRhodamine (xR) attached to the ends of a 29-mer duplex DNA.