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Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers

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Molecular Motors

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

Abstract

Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a preformed prohead shell in ~5 min at room temperature. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers. Three viral packaging systems are described in detail: bacteriophages phi29 (φ29), lambda (λ), and T4. Two different approaches are described: (1) With φ29 and T4, prohead–motor complexes can be preassembled in bulk and packaging can be initiated in the optical tweezers by “feeding” a single DNA molecule to one of the complexes; (2) With φ29 and λ, packaging can be initiated in bulk then stalled, and a single prohead–motor–DNA complex can then be captured with optical tweezers and restarted. In both cases, the prohead is ultimately attached to one trapped microsphere and the end of the DNA being packaged is attached to a second trapped microsphere such that packaging of the DNA pulls the two microspheres together and the rate of packaging and force generated by the motor is directly measured in real time. These protocols allow for the effect of many experimental parameters on packaging dynamics to be studied such as temperature, ATP concentration, ionic conditions, structural changes to the DNA substrate, and mutations in the motor proteins. Procedures for capturing microspheres with the optical traps and different measurement modes are also described.

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Acknowledgments

We thank Dwight Anderson, Carlos Bustamante, Carlos Catalano, Yann Chemla, Michael Feiss, Derek Fuller, Shelley Grimes, Paul Jardine, Vishal Kottadiel, Shixin Liu, Jeff Moffitt, Mariam Ordyan, Venigalla Rao, John Peter Rickgauer, Jean Sippy, Steven Smith, Sander Tans, and James Tsay for assistance. This work was supported, in part, by NSF awards PHY-0848905 and MCB-1158328 and NIH award R01-GM088186.

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Authors and Affiliations

  1. Department of Physics, University of California San Diego, La Jolla, CA, USA

    Nicholas Keller, Damian J. delToro & Douglas E. Smith

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  1. Nicholas Keller
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  2. Damian J. delToro
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  3. Douglas E. Smith
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Correspondence to Douglas E. Smith .

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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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Keller, N., delToro, D.J., Smith, D.E. (2018). Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers. 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_20

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  • DOI: https://doi.org/10.1007/978-1-4939-8556-2_20

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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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