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Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution

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Book cover Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1486))

Abstract

During transcriptional elongation, RNA polymerases (RNAP) employ a stepping mechanism to translocate along the DNA template while synthesizing RNA. Optical trapping assays permit the progress of single molecules of RNA polymerase to be monitored in real time, at resolutions down to the level of individual base pairs. Additionally, optical trapping assays permit the application of exquisitely controlled, external forces on RNAP. Responses to such forces can reveal details of the load-dependent kinetics of transcriptional elongation and pausing. Traditionally, the bacterial form of RNAP from E. coli has served as a model for the study of transcriptional elongation using optical traps. However, it is now feasible to perform optical trapping experiments using the eukaryotic polymerase, RNAPII, as well. In this report, we describe the methods to perform optical trapping transcriptional elongation assays with both prokaryotic RNAP and eukaryotic RNAPII. We provide detailed instructions on how to reconstitute transcription elongation complexes, derivatize beads used in the assays, and perform optical trapping measurements.

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Author information

Author notes

    Authors and Affiliations

    1. Department of Biology, Stanford University, Stanford, CA, USA

      Anirban Chakraborty

    2. Department of Chemistry, Stanford University, Stanford, CA, USA

      Cong A. Meng

    3. Department of Biology and Department of Applied Physics, Stanford University, Stanford, CA, USA

      Steven M. Block

    Authors
    1. Anirban Chakraborty
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    2. Cong A. Meng
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    3. Steven M. Block
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    Corresponding author

    Correspondence to Steven M. Block .

    Editor information

    Editors and Affiliations

    1. Department of Anatomy and Structural Biology & Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

      Arne Gennerich

    1 Electronic Supplementary Material

    Below is the link to the electronic supplementary material.

    Capturing and testing a dumbbell with optical tweezers (WMV 14,606 kB).

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    Chakraborty, A., Meng, C.A., Block, S.M. (2017). Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 1486. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6421-5_15

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    • DOI: https://doi.org/10.1007/978-1-4939-6421-5_15

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6419-2

    • Online ISBN: 978-1-4939-6421-5

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