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Using Solutes and Kinetics to Probe Large Conformational Changes in the Steps of Transcription Initiation

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Bacterial Transcriptional Control

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1276))

Abstract

Small solutes are useful probes of large conformational changes in RNA polymerase–promoter interactions and other biopolymer processes. In general, a large effect of a solute on an equilibrium constant (or rate constant) indicates a large change in water-accessible biopolymer surface area in the corresponding step (or transition state), resulting from conformational changes, interface formation, or both. Here, we describe nitrocellulose filter binding assays from series used to determine the urea dependence of open complex formation and dissociation with Escherichia coli RNA polymerase and phage λPR promoter DNA. Then, we describe the subsequent data analysis and interpretation of these solute effects.

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Acknowledgements

Research from our laboratory reviewed here is supported by NIH GM103061.

Author information

Authors and Affiliations

  1. Department of Chemistry, 3206 BSB, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Emily F. Ruff & M. Thomas Record Jr.

  2. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Wayne S. Kontur

  3. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA

    M. Thomas Record Jr.

Authors
  1. Emily F. Ruff
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  2. Wayne S. Kontur
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  3. M. Thomas Record Jr.
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Corresponding authors

Correspondence to Emily F. Ruff or M. Thomas Record Jr. .

Editor information

Editors and Affiliations

  1. Department of Microbiology, The Ohio State University, Columbus, Ohio, USA

    Irina Artsimovitch

  2. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA

    Thomas J. Santangelo

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Ruff, E.F., Kontur, W.S., Record, M.T. (2015). Using Solutes and Kinetics to Probe Large Conformational Changes in the Steps of Transcription Initiation. In: Artsimovitch, I., Santangelo, T. (eds) Bacterial Transcriptional Control. Methods in Molecular Biology, vol 1276. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2392-2_14

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  • DOI: https://doi.org/10.1007/978-1-4939-2392-2_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2391-5

  • Online ISBN: 978-1-4939-2392-2

  • eBook Packages: Springer Protocols

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