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Single-Molecule Angular Optical Trapping for Studying Transcription Under Torsion

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Molecular Motors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1805))

Abstract

Optical tweezers are flexible and powerful single-molecule tools that have been extensively utilized in biophysical studies. With their ability to stretch and twist DNA, and measure its force and torque simultaneously, they provide excellent opportunities to gain novel insights into the function of protein motors and protein-DNA interactions. Recently, a novel DNA supercoiling assay using an angular optical tweezers (AOT) has been developed to investigate torque generation during transcription. Here, we provide a detailed and practical guide to performing this technique. Using bacterial RNA polymerase (RNAP) as an example, we present protocols for constructing and calibrating an AOT instrument, preparing DNA templates, and acquiring and analyzing real-time data for transcription under DNA supercoiling. While these protocols were initially developed with E. coli RNAP, they can be readily adapted to study other DNA-based motor proteins.

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Acknowledgments

We thank Shanna Moore for commenting on this manuscript and Drs. Scott Forth, James Inman and Maxim Sheinin for the discussion and help. This work was supported by the Howard Hughes Medical Institute (to M.D.W.), the National Science Foundation grants (MCB-0820293 and MCB-1517764 to M.D.W.), the National Natural Science Foundation of China (NSFC-11674403 to J.M.), and the Fundamental Research Funds for the Central Universities (15lgjc15 to J.M.).

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Authors and Affiliations

  1. School of Physics, Sun Yat-sen University, Guangzhou, People’s Republic of China

    Jie Ma

  2. Laboratory of Atomic and Solid State Physics, Department of Physics, Cornell University, Ithaca, NY, USA

    Chuang Tan & Michelle D. Wang

  3. Howard Hughes Medical Institute, Cornell University, Ithaca, NY, USA

    Chuang Tan & Michelle D. Wang

Authors
  1. Jie Ma
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  2. Chuang Tan
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  3. Michelle D. Wang
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Corresponding author

Correspondence to Michelle D. Wang .

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Editors and Affiliations

  1. National Museum of Natural History, CNRS UMR7196 / INSERM U1154, Paris, France

    Christophe Lavelle

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Ma, J., Tan, C., Wang, M.D. (2018). Single-Molecule Angular Optical Trapping for Studying Transcription Under Torsion. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_16

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  • DOI: https://doi.org/10.1007/978-1-4939-8556-2_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8554-8

  • Online ISBN: 978-1-4939-8556-2

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