Intersubunit coordination in a homomeric ring ATPase


Homomeric ring ATPases perform many vital and varied tasks in the cell, ranging from chromosome segregation to protein degradation. Here we report the direct observation of the intersubunit coordination and step size of such a ring ATPase, the double-stranded-DNA packaging motor in the bacteriophage ϕ29. Using high-resolution optical tweezers, we find that packaging occurs in increments of 10 base pairs (bp). Statistical analysis of the preceding dwell times reveals that multiple ATPs bind during each dwell, and application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps. These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated by means of a mechanism novel for ring ATPases. Furthermore, a step size that is a non-integer number of base pairs demands new models for motor–DNA interactions.

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Figure 1: Bacteriophage ϕ29 packages DNA in bursts of 10 bp.
Figure 2: Dwells before 10-bp bursts contain multiple kinetic events.
Figure 3: The 10-bp bursts are composed of four 2.5-bp steps.
Figure 4: Intersubunit coordination in the ring ATPase of ϕ29.
Figure 5: Packaging models that produce a non-integer step size.


  1. 1

    Latterich, M. & Patel, S. The AAA team: related ATPases with diverse functions. Trends Cell Biol. 8, 65–71 (1998)

    Article  Google Scholar 

  2. 2

    Ogura, T. & Wilkinson, A. J. AAA+ superfamily ATPases: common structure–diverse function. Genes Cells 6, 575–597 (2001)

    CAS  Article  Google Scholar 

  3. 3

    Iyer, L. M., Leipe, D. D., Koonin, E. V. & Aravind, L. Evolutionary history and higher order classification of AAA+ ATPases. J. Struct. Biol. 146, 11–31 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Kainov, D. E., Tuma, R. & Mancini, E. J. Hexameric molecular motors: P4 packaging ATPase unravels the mechanism. Cell. Mol. Life Sci. 63, 1095–1105 (2006)

    CAS  Article  Google Scholar 

  5. 5

    Erzberger, J. P. & Berger, J. M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 93–114 (2006)

    CAS  Article  Google Scholar 

  6. 6

    Singleton, M. R., Sawaya, M. R., Ellenberger, T. & Wigley, D. B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000)

    CAS  Article  Google Scholar 

  7. 7

    Mancini, E. J. et al. Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell 118, 743–755 (2004)

    CAS  Article  Google Scholar 

  8. 8

    Kinosita, K., Adachi, K. & Itoh, H. Rotation of F1-ATPase: How an ATP-driven molecular machine may work. Annu. Rev. Biophys. Biomol. Struct. 33, 245–268 (2004)

    CAS  Article  Google Scholar 

  9. 9

    Enemark, E. J. & Joshua-Tor, L. Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442, 270–275 (2006)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Skordalakes, E. & Berger, J. M. Structural insights into RNA-dependent ring closure and ATPase activation by the Rho termination factor. Cell 127, 553–564 (2006)

    CAS  Article  Google Scholar 

  11. 11

    Adelman, J. L. et al. Mechanochemistry of transcription termination factor Rho. Mol. Cell 22, 611–621 (2006)

    CAS  Article  Google Scholar 

  12. 12

    Liao, J.-C., Jeong, Y.-J., Kim, D.-E., Patel, S. S. & Oster, G. Mechanochemistry of T7 DNA helicase. J. Mol. Biol. 350, 452–475 (2005)

    CAS  Article  Google Scholar 

  13. 13

    Massey, T. H., Mercogliano, C. P., Yates, J., Sherratt, D. J. & Löwe, J. Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol. Cell 23, 457–469 (2006)

    CAS  Article  Google Scholar 

  14. 14

    Crampton, D. J., Mukherjee, S. & Richardson, C. C. DNA-induced switch from independent to sequential dTTP hydrolysis in the bacteriophage T7 DNA helicase. Mol. Cell 21, 165–174 (2006)

    CAS  Article  Google Scholar 

  15. 15

    Gai, D., Zhao, R., Li, D., Finkielstein, C. V. & Chen, X. S. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen. Cell 119, 47–60 (2004)

    CAS  Article  Google Scholar 

  16. 16

    Martin, A., Baker, T. A. & Sauer, R. T. Rebuilt AAA+ motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115–1120 (2005)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Guo, P., Grimes, S. & Anderson, D. A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage ϕ29. Proc. Natl Acad. Sci. USA 83, 3505–3509 (1986)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Smith, D. E. et al. The bacteriophage straight ϕ29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Chemla, Y. R. et al. Mechanism of force generation of a viral DNA packaging motor. Cell 122, 683–692 (2005)

    CAS  Article  Google Scholar 

  20. 20

    Grimes, S., Jardine, P. J. & Anderson, D. Bacteriophage ϕ29 DNA packaging. Adv. Virus Res. 58, 255–294 (2002)

    CAS  Article  Google Scholar 

  21. 21

    Hugel, T. et al. Experimental test of connector rotation during DNA packaging into bacteriophage ϕ29 capsids. PLoS Biol. 5, e59 (2007)

    Article  Google Scholar 

  22. 22

    Fuller, D. N. et al. Ionic effects on viral DNA packaging and portal motor function in bacteriophage ϕ29. Proc. Natl Acad. Sci. USA 104, 11245–11250 (2007)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Rickgauer, J. P. et al. Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage ϕ29. Biophys. J. 94, 159–167 (2008)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Simpson, A. A. et al. Structure of the bacteriophage ϕ29 DNA packaging motor. Nature 408, 745–750 (2000)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Morais, M. C. et al. Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage ϕ29. J. Struct. Biol. 135, 38–46 (2001)

    CAS  Article  Google Scholar 

  26. 26

    Morais, M. C. et al. Defining molecular and domain boundaries in the bacteriophage ϕ29 DNA packaging motor. Structure 16, 1267–1274 (2008)

    CAS  Article  Google Scholar 

  27. 27

    Burroughs, A. M., Iyer, L. M. & Aravind, L. in Gene and Protein Evolution (ed. Volff, J.-N.) 48–65 (Karger, 2007)

    Google Scholar 

  28. 28

    Iyer, L. M., Makarova, K. S., Koonin, E. V. & Aravind, L. Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging. Nucleic Acids Res. 32, 5260–5279 (2004)

    CAS  Article  Google Scholar 

  29. 29

    Guo, P., Peterson, C. & Anderson, D. Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage ϕ29. J. Mol. Biol. 197, 229–236 (1987)

    CAS  Article  Google Scholar 

  30. 30

    Chen, C. & Guo, P. Sequential action of six virus-encoded DNA-packaging RNAs during phage ϕ29 genomic DNA translocation. J. Virol. 71, 3864–3871 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Moffitt, J. R., Chemla, Y. R., Smith, S. B. & Bustamante, C. Recent advances in optical tweezers. Annu. Rev. Biochem. 77, 205–228 (2008)

    CAS  Article  Google Scholar 

  32. 32

    Moffitt, J. R., Chemla, Y. R., Izhaky, D. & Bustamante, C. Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc. Natl Acad. Sci. USA 103, 9006–9011 (2006)

    ADS  CAS  Article  Google Scholar 

  33. 33

    Bustamante, C., Chemla, Y. R. & Moffitt, J. R. in Single-Molecule Techniques: A Laboratory Manual (eds Selvin, P. R. & Ha, T.) 297–324 (Cold Spring Harbor Laboratories, 2008)

    Google Scholar 

  34. 34

    Schnitzer, M. J. & Block, S. M. Statistical kinetics of processive enzymes. Cold Spring Harb. Symp. Quant. Biol. 60, 793–802 (1995)

    CAS  Article  Google Scholar 

  35. 35

    Koza, Z. Maximal force exerted by a molecular motor. Phys. Rev. E 65, 031905 (2002)

    ADS  Article  Google Scholar 

  36. 36

    Chemla, Y. R., Moffitt, J. R. & Bustamante, C. Exact solutions for kinetic models of macromolecular dynamics. J. Phys. Chem. B 112, 6025–6044 (2008)

    CAS  Article  Google Scholar 

  37. 37

    Bustamante, C., Chemla, Y. R., Forde, N. R. & Izhaky, D. Mechanical processes in biochemistry. Annu. Rev. Biochem. 73, 705–748 (2004)

    CAS  Article  Google Scholar 

  38. 38

    Segel, I. H. Enzyme Kinetics (John Wiley & Sons, 1975)

    Google Scholar 

  39. 39

    Oster, G. & Wang, H. Reverse engineering a protein: the mechanochemistry of ATP synthase. Biochim. Biophys. Acta 1458, 482–510 (2000)

    CAS  Article  Google Scholar 

  40. 40

    Skordalakes, E. & Berger, J. M. Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell 114, 135–146 (2003)

    CAS  Article  Google Scholar 

  41. 41

    Lisal, J. et al. Functional visualization of viral molecular motor by hydrogen-deuterium exchange reveals transient states. Nature Struct. Mol. Biol. 12, 460–466 (2005)

    CAS  Article  Google Scholar 

  42. 42

    Moreau, M. J., McGeoch, A. T., Lowe, A. R., Itzhaki, L. S. & Bell, S. D. ATPase site architecture and helicase mechanism of an archaeal MCM. Mol. Cell 28, 304–314 (2007)

    CAS  Article  Google Scholar 

  43. 43

    Berg-Sorensen, K. & Flyvbjerg, H. Power spectrum analysis for optical tweezers. Rev. Sci. Instrum. 75, 594–612 (2004)

    ADS  CAS  Article  Google Scholar 

  44. 44

    Block, S. M. & Svoboda, K. Analysis of high resolution recordings of motor movement. Biophys. J. 68, 230–241 (1995)

    Google Scholar 

  45. 45

    Carter, N. J. & Cross, R. A. Mechanics of the kinesin step. Nature 435, 308–312 (2005)

    ADS  CAS  Article  Google Scholar 

  46. 46

    Parzen, E. On estimation of a probability density function and mode. Ann. Math. Stat. 33, 1065–1076 (1962)

    MathSciNet  Article  Google Scholar 

  47. 47

    Grimes, S. & Anderson, D. The bacteriophage ϕ29 packaging proteins supercoil the DNA ends. J. Mol. Biol. 266, 901–914 (1997)

    CAS  Article  Google Scholar 

  48. 48

    Kellner, L. The near infra-red absorption spectrum of heavy water. Proc. R. Soc. Lond. A 159, 0410–0415 (1937)

    ADS  CAS  Article  Google Scholar 

  49. 49

    Baumann, C. G., Smith, S. B., Bloomfield, V. A. & Bustamante, C. Ionic effects on the elasticity of single DNA molecules. Proc. Natl Acad. Sci. USA 94, 6185–6190 (1997)

    ADS  CAS  Article  Google Scholar 

  50. 50

    Yanagi, K., Prive, G. G. & Dickerson, R. E. Analysis of local helix geometry in three B-DNA decamers and eight dodecamers. J. Mol. Biol. 217, 201–214 (1991)

    CAS  Article  Google Scholar 

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We thank C. L. Hetherington, M. Nollmann and G. Chistol for a critical reading of the manuscript; C. L. Hetherington, A. Politzer, M. Strycharska, M. Kopaczynska and J. Yu for critical discussions; and J. Choy, S. Grill and S. Smith for advice regarding instrumentation. J.R.M. acknowledges the National Science Foundation’s Graduate Research Fellowship and Y.R.C. the Burroughs Welcome Fund’s Career Awards at the Scientific Interface for funding. This research was supported in part by NIH grants GM-071552, DE-003606 and GM-059604. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author Contributions J.R.M., Y.R.C. and K.A. conducted the experiments and performed the analysis; S.G., P.J.J. and D.L.A. prepared and provided experimental materials; and J.R.M., Y.R.C., K.A., S.G., P.J.J., D.L.A. and C.B. wrote the paper. J.R.M. and Y.R.C. contributed equally to this work.

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Correspondence to Carlos Bustamante.

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Moffitt, J., Chemla, Y., Aathavan, K. et al. Intersubunit coordination in a homomeric ring ATPase. Nature 457, 446–450 (2009).

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