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Viral DNA Packaging: One Step at a Time

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Single Molecule Spectroscopy in Chemistry, Physics and Biology

Part of the book series: Springer Series in Chemical Physics ((CHEMICAL,volume 96))

Summary

During its life-cycle the bacteriophage φ29 actively packages its dsDNA genome into a proteinacious capsid, compressing its genome to near crystalline densities against large electrostatic, elastic, and entropic forces. This remarkable process is accomplished by a nano-scale, molecular DNA pump – a complex assembly of three protein and nucleic acid rings which utilizes the free energy released in ATP hydrolysis to perform the mechanical work necessary to overcome these large energetic barriers. We have developed a single molecule optical tweezers assay which has allowed us to probe the detailed mechanism of this packaging motor. By following the rate of packaging of a single bacteriophage as the capsid is filled with genome and as a function of optically applied load, we find that the compression of the genome results in the build-up of an internal force, on the order of ∼ 55 pN, due to the compressed genome. The ability to work against such large forces makes the packaging motor one of the strongest known molecular motors. By titrating the concentration of ATP, ADP, and inorganic phosphate at different opposing load, we are able to determine features of the mechanochemistry of this motor – the coupling between the mechanical and chemical cycles. We find that force is generated not upon binding of ATP, but rather upon release of hydrolysis products. Finally, by improving the resolution of the optical tweezers assay, we are able to observe the discrete increments of DNA encapsidated each cycle of the packaging motor. We find that DNA is packaged in 10-bp increments preceded by the binding of multiple ATPs. The application of large external forces slows the packaging rate of the motor, revealing that the 10-bp steps are actually composed of four 2.5-bp steps which occur in rapid succession. These data show that the individual subunits of the pentameric ring-ATPase at the core of the packaging motor are highly coordinated, with the binding of ATP and the translocation of DNA temporally segregated into two distinct phases of the mechanochemical cycle of the entire ring. Because this ring-ATPase is a member of the ASCE superfamily of ATPases, these results may have implications for a broad and diverse family of cellular motors.

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Acknowledgements

J.R.M acknowledges the National Science Foundation’s Graduate Research Fellowship for support. This work was supported in part by NIH grants GM-071552, DE-003606, and GM-059604, and DOE grant DE-AC03–76DF00098.

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Authors and Affiliations

  1. Jason L. Choy Laboratory of Single Molecule Biophysics and Department of Physics, University of California, Berkeley, CA, 94720, USA

    Carlos Bustamante & Jeffrey R. Moffitt

  2. Departments of Chemistry and Molecular and Cell Biology Howard Hughes Medical Institute, University of California, Berkeley, CA, 94720, USA

    Carlos Bustamante

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  1. Carlos Bustamante
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Correspondence to Carlos Bustamante .

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Editors and Affiliations

  1. Dept. Biophysics, Stockholm University, Stockholm, 106 91, Sweden

    Astrid Gräslund

  2. rue du Centre 115, St. Sulpice, 1025, Switzerland

    Rudolf Rigler

  3. Dept. Biomolecular Physics, Royal Institute of Technology (KTH), Stockholm, 106 91, Sweden

    Jerker Widengren

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Bustamante, C., Moffitt, J.R. (2010). Viral DNA Packaging: One Step at a Time. In: Gräslund, A., Rigler, R., Widengren, J. (eds) Single Molecule Spectroscopy in Chemistry, Physics and Biology. Springer Series in Chemical Physics, vol 96. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-02597-6_13

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