Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Thomas T Perkins

Second Advisor

James Goodrich

Third Advisor

Matthew Glaser

Abstract

TATA-Binding Protein (TBP) binding to DNA is a critical step in gene expression. In the first step of gene expression, transcription of RNA from DNA, TBP binds DNA and recruits the transcription machinery. TBP binds DNA at a preferred sequence called the TATA box and bends the DNA at ~100°. In this work we characterized TBP dynamically bending DNA at the single-molecule level using an actively stabilized, axial optical trap.

To perform this characterization, several challenges led us to develop a customized biophysical assay. Nonspecific interactions of TBP with sample surfaces required coating surfaces with polyethylene glycol. A small predicted signal size (~5 nm) and slow kinetics (~10-2 s-1) demanded high stability in our optical trap assay, leading us to implement an actively stabilized instrument. An apparent affnity by TBP for non-TATA box DNA sequences led us to use very short DNA molecules (92 nm) with carefully controlled sequences. This short length of DNA demanded the development of a novel axial trapping and detection technique, which also has the advantage of improved spatiotemporal resolution (integrated noise <1 nm over 100 s at 0.5 pN, 5 fN/nm trap stiffness, Δf = 0.03-3.2 Hz). Our final assay involved custom surface-chemistry, an actively stabilized optical trap, short DNA with carefully engineered sequences, and a novel axial detection method.

Using this novel assay, we measured TBP and TATA-box dependent extension changes of DNA at the single-molecule level. Under optimized conditions (short, carefully chosen DNA; optimized [TBP], [MgCl2] and [KCl]), we obtained step-wise, consistently-sized TBP- dependent extension changes. By hidden Markov modeling analysis, we quantified the extension changes and rates for bending and unbending, and we performed simulations to verify our analysis methods. We applied three different forces (0.3, 0.5 and 0.8 pN) to test the affect of force on the extension change and rates. We found a constant extension change of 3 nm, with dynamics on the scale of tens of seconds. The developed assay directly measures dynamic bending of DNA by TBP, and the techniques developed here have potential to be broadly useful for high-spatiotemporal resolution studies of many other DNA-protein interactions.

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