Date of Award

Spring 1-1-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Jennifer F. Kugel

Second Advisor

Amy Palmer

Third Advisor

Arthur Pardi

Fourth Advisor

Thomas Perkins

Fifth Advisor

Robert Kuchta

Abstract

Defining mechanisms of transcriptional regulation is important for understanding how gene expression is controlled, which is essential to cellular viability. Outlined in this thesis are studies that characterize DNA-protein interactions involved in transcriptional regulation. Specifically, we have used single-molecule FRET (smFRET) to investigate how proteins bend DNA. A homebuilt TIRF microscope was assembled for single molecule fluorescence studies, which is described in Chapter 2 of this thesis.

We used smFRET to study the extent and kinetics of DNA bending by the transcription factor TBP (TATA binding protein) with consensus and nonconsensus TATA DNA. TBP bent different TATA sequences to the same, homogenous, bent population. Further, TFIIA did not change the extent of DNA bending by TBP, but increased the stability of the TBP-DNA complex. We found that TBP bent consensus TATA DNA to two different kinetic populations bent to the same extent, but only one kinetic bent population existed when TBP was bound to the nonconsensus sequence. The uniform bending of DNA by TBP was not predicted by previous ensemble studies and provided insight into the mechanism of DNA binding by TBP.

HMGB1 is a nuclear protein that binds and bends DNA to facilitate transcription. It has 3 domains: the A and B box, which bend DNA independently, and an acidic unstructured C-terminal tail. We studied how full length HMGB1 and its different domains bend DNA using smFRET. We determined that the full length HMGB1 protein bent DNA to a similar extent as its individual domains, the A box and the B box. However, removal of the C-terminal tail caused the protein to bend DNA to a greater extent and with more heterogeneity than the full length protein. Further, a truncation that contains the B box and C-terminal tail interacted with DNA less efficiently than the B box alone; this truncation could bend DNA at a high concentration to the same extent as the B box alone. Taken together we propose that the full length HMGB1 protein bends DNA primarily through its A box, while the B box interacts with the C-terminal tail, which prevents its interaction with DNA.

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