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Biophysical studies on multiple DNA duplexes and on a protein·protein·DNA transcription complex
Abstract
Time-resolved fluorescence emission measurements, in conjunction with fluorescence resonance energy transfer (FRET), were used to determine the two parameters for a Gaussian inter-dye distance distribution, R¯ and σ, for a series of eleven DNA duplexes from 11 to 21 bp in length. Dyes are linked by flexible tethers to DNA to achieve an averaging of FRET dipolar interactions by rapid rotational motion of the dyes. Steady-state and time-resolved fluorescence emission anisotropy measurements have been used to establish limits on the value of κ2 for our donor/acceptor FRET pair. Our eleven duplexes have the smallest values for σ that we have measured, and correspond to straight and rigid duplexes, with a reflecting the dye-DNA distance distribution. We have fit all of the FRET data as a set to obtain the average positions and motions of the donor and acceptor dyes. The results indicate that this particular dye pair gives average inter-dye distances that are not strongly dependent on dye phasing, allowing simple measurements of DNA bending to be made. TheR¯ and σ values from the stiff and straight oligonucleotides will in turn allow both static bends and flexibility to be determined for other oligonucleotides employing this dye pair. We have also selectively modified DNA using one or two centrally located deoxyuracils substituted at the 5-position with either a flexible 3-aminopropyl group or a rigid aminopropynyl group. R¯ and σ were obtained from time-resolved FRET measurements. Duplex flexibility is altered by the presence of a single positive charge. In contrast, the mean end-to-end distance is shown to be significantly altered by the introduction of two adjacently tethered cations into the double helix but not by a single cation. These changes in the helical structure are attributed to selective perturbation of the electrostatic potential of the helix around the cation(s). Finally, we have been studying the kinetics and thermodynamics of the first steps in the assembly of the eukaryotic transcription complex formed from TBP (TATA Binding Protein), DNA, and TFIIA (Transcription Factor IIA). Stopped-flow anisotropy studies show TFIIA binds tightly and within a few seconds to TBP bound to native TATA boxes.
Subject Area
Biochemistry
Recommended Citation
Williams, Sarah L, "Biophysical studies on multiple DNA duplexes and on a protein·protein·DNA transcription complex" (2006). ETD collection for University of Nebraska-Lincoln. AAI3214725.
https://digitalcommons.unl.edu/dissertations/AAI3214725