Deciphering the Molecular Mechanism of the Bacteriophage φ29 DNA Packaging Motor

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

Abstract

The past decade has seen an explosion in the use of single-molecule approaches to study complex biological processes. One such approach—optical trapping—is particularly well suited for investigating molecular motors, a diverse group of macromolecular complexes that convert chemical energy into mechanical work, thus playing key roles in virtually every aspect of cellular life. Here we describe how to use high-resolution optical tweezers to investigate the mechanism of the bacteriophage φ29 DNA packaging motor, a ring-shaped ATPase responsible for genome packing during viral assembly. This system illustrates how to use single-molecule techniques to uncover novel, often unexpected, principles of motor operation.

Key words

Single-molecule manipulation Optical tweezers Viral DNA packaging Molecular motor Ring ATPase 

References

  1. 1.
    Hetherington CL, Moffitt JR, Jardine PJ et al (2012) Viral DNA packaging motors. In: Goldman YE, Ostap EM (eds) Molecular motors and motility. Elsevier, Oxford, pp 420–446Google Scholar
  2. 2.
    Morais MC (2012) The dsDNA packaging motor in bacteriophage φ29. In: Rossmann MG, Rao VB (eds) Viral molecular machines. Springer, New York, NY, pp 511–547CrossRefGoogle Scholar
  3. 3.
    Smith DE, Tans SJ, Smith SB et al (2001) The bacteriophage φ29 portal motor can package DNA against a large internal force. Nature 413:748–752CrossRefGoogle Scholar
  4. 4.
    Chemla YR, Aathavan K, Michaelis J et al (2005) Mechanism of force generation of a viral DNA packaging motor. Cell 122:683–692CrossRefGoogle Scholar
  5. 5.
    Moffitt JR, Chemla YR, Aathavan K et al (2009) Intersubunit coordination in a homomeric ring ATPase. Nature 457:446–450CrossRefGoogle Scholar
  6. 6.
    Aathavan K, Politzer AT, Kaplan A et al (2009) Substrate interactions and promiscuity in a viral DNA packaging motor. Nature 461:669–673CrossRefGoogle Scholar
  7. 7.
    Chistol G, Liu S, Hetherington CL et al (2012) High degree of coordination and division of labor among subunits in a homomeric ring ATPase. Cell 151:1017–1028CrossRefGoogle Scholar
  8. 8.
    Liu S, Chistol G, Hetherington CL et al (2014) A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills. Cell 157:702–713CrossRefGoogle Scholar
  9. 9.
    Liu S, Chistol G, Bustamante C (2014) Mechanical operation and intersubunit coordination of ring-shaped molecular motors: insights from single-molecule studies. Biophys J 106:1844–1858CrossRefGoogle Scholar
  10. 10.
    Zhao W, Morais MC, Anderson DL et al (2008) Role of the CCA bulge of prohead RNA of bacteriophage φ29 in DNA packaging. J Mol Biol 383:520–528CrossRefGoogle Scholar
  11. 11.
    Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75:2787–2809CrossRefGoogle Scholar
  12. 12.
    Berg-Sørensen K, Flyvbjerg H (2004) Power spectrum analysis for optical tweezers. Rev Sci Instrum 75:594–612CrossRefGoogle Scholar
  13. 13.
    Bustamante C, Marko JF, Siggia ED et al (1994) Entropic elasticity of lambda-phage DNA. Science 265:1599–1600CrossRefGoogle Scholar
  14. 14.
    Bustamante C, Chemla YR, Moffitt JR (2008) High-resolution dual-trap optical tweezers with differential detection. In: Selvin PR, Ha T (eds) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 297–324Google Scholar
  15. 15.
    Grimes S, Jardine PJ, Anderson D (2002) Bacteriophage φ29 DNA packaging. Adv Virus Res 58:255–294CrossRefGoogle Scholar
  16. 16.
    Rickgauer JP, Fuller DN, Grimes S et al (2008) Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage φ29. Biophys J 94:159–167CrossRefGoogle Scholar
  17. 17.
    Kalafut B, Visscher K (2008) An objective, model-independent method for detection of non-uniform steps in noisy signals. Comput Phys Commun 179:716–723CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.The Rockefeller UniversityNew YorkUSA
  2. 2.University of CaliforniaBerkeleyBerkeleyUSA
  3. 3.University of California and Howard Hughes Medical InstituteBerkeleyBerkeleyUSA

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