Bioanalytical Methods Development for the Study of Regulation in Transcription Elongation Public Deposited

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  • March 20, 2019
  • Brodnick, Robert Daniel
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • Previous biochemical data has shown that transcription by multisubunit RNA polymerases is heavily regulated by the interactions between the ternary elongation complex and catalytic and non-catalytic templated NTPs. While kinetics studies and crystal structures have aided in the generation of mechanistic models for nucleotide incorporation with stalled elongation complexes (SEC), quantitative observation of NTP-SEC binding properties has not been achieved. The primary limitation in NTP-SEC binding analysis is the lack available methods capable of measuring binding when only low nanomolar amounts of protein acceptor (in this case, the SECs) are available. In this work, I developed a number of purification techniques for the study of NTP-SEC binding, including novel approaches in electrodialysis, microfiber dialysis, gel electrophoresis and other phase separations. I report that some of these methods show promise for universal application in small molecule-protein binding assays. One method termed reversible matrix assisted phase partitioning (RevMAPP) facilitated the direct capture of NTP-SEC occupancies (stoichiometries), and many other binding properties. I was able to increase the signal to noise ratio in radiochemical binding analyses by using a monomeric avidin coated matrix to synthesize and purify biotinalated SECs. I have determined that subsequent to SEC purification, a high occupancy of non-templated nucleotides remain bound to the enzyme, exhibiting very slow passive (non-competitive) rates of dissociation. Parts of our previous mechanistic models for regulation in transcription elongation by allosteric NTP binding will need minor adjustments to fit these new data. In addition to NTP-SEC binding studies and method development, I present a myriad of glass surface preparation protocols for the purpose of conducting atomic force microscopy on multimeric biological complexes. Conducting biological AFM on glass will streamline the combination of high resolution single molecule fluorescence (SMF) techniques with AFM to bolster the structure-function information we are capable of currently capturing with the each microscopy technique by itself. Molecular alignment on DNA on glass is the primary limitation in conducting structure-function AFM studies. For AFM imaging on glass, I present a minimal force deposition method for preparing DNA samples in a way that does not stretch or align the DNA molecules. I present the first high resolution AFM images of protein-DNA complexes deposited onto smoothed glass under physiological conditions.
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  • Erie, Dorothy
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