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Manipulating Archaeal Systems to Permit Analyses of Transcription Elongation-Termination Decisions In Vitro

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Bacterial Transcriptional Control

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

Abstract

Transcription elongation by multisubunit RNA polymerases (RNAPs) is processive, but neither uniform nor continuous. Regulatory events during elongation include pausing, backtracking, arrest, and transcription termination, and it is critical to determine whether the absence of continued synthesis is transient or permanent. Here we describe mechanisms to generate large quantities of stable archaeal elongation complexes on a solid support to permit (1) single-round transcription, (2) walking of RNAP to any defined template position, and (3) discrimination of transcripts that are associated with RNAP from those that are released to solution. This methodology is based on untagged proteins transcribing biotin- and digoxigenin-labeled DNA templates in association with paramagnetic particles.

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Acknowledgements

This work was supported by a grant (GM-100329) from the National Institutes of Health to TJS.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, 383 MRB, Colorado State University, Fort Collins, CO, 80523, USA

    Alexandra M. Gehring & Thomas J. Santangelo

Authors
  1. Alexandra M. Gehring
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  2. Thomas J. Santangelo
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Corresponding author

Correspondence to Thomas J. Santangelo .

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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Gehring, A.M., Santangelo, T.J. (2015). Manipulating Archaeal Systems to Permit Analyses of Transcription Elongation-Termination Decisions In Vitro. 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_15

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  • DOI: https://doi.org/10.1007/978-1-4939-2392-2_15

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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

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