Regulation of transcription elongation via interactions of RNA polymerase with sequence elements and accessory factors Public Deposited

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  • March 21, 2019
  • Pearson, Erika L.
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • RNA polymerase (RNAP) is the enzyme responsible for catalyzing the first step in a cascade of events leading towards gene expression. Since the dissemination of genetic information plays such a critical and integral role within the context of cellular processes, RNAP must uphold the stringent guidelines of catalyzing processive DNA-directed synthesis of RNA transcripts at a reasonable rate and with high fidelity. To ensure that these requirements are met and sustained, RNAP serves as a major target of rigorous regulation and is consequently subject to extrinsic regulatory factors, such as transcription accessory proteins, and to regulatory sequence elements, such as pause and termination sites located throughout the genome. Chapter 2 explores the ability of RNAP to recognize a terminally misincorporated base in the nascent RNA transcript and to cleave that base from the transcript. Additionally, the effects of GreA and GreB, transcript cleavage factors, on RNAP containing terminally misincorporated transcript are also investigated. My results indicate that RNAP does not preferentially cleave a misincorporated base. In fact, the GreB-mediated cleavage of terminally correct and terminally incorrect bases is indistinguishable. I conclude that, in the event of misincorporation, decay into the activated pathway serves as a signal to recruit GreA and GreB. Through Gre-mediated action, RNAP may backtrack along the nascent transcript and cleave the segment of RNA containing the error. Chapter 3 studies the role of downstream elements in RNAP in regulating transcription elongation. RNAPs harboring mutations in two regions (Walker B motif and fork loop 2) composing the allosteric NTP binding site are studied for their ability to recognize pause and termination sites. I demonstrate that the Walker B motif is important for the recognition of pause and termination sites. The recognition of termination signals by the Walker B motif is also important in vivo. Additionally, I propose that the formation of two classes of pause states does not proceed through a common slow intermediate, as previously thought. Chapter 4 presents the development of method by which the interaction of a transcription accessory factor with a stalled elongation complex can be probed. This method utilizes the technique of through-prism total internal reflection fluorescence (TIRF) microscopy. I present single-molecule data that permit me to estimate the lifetime and the dissociation constant of NusG.
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  • Erie, Dorothy
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