Abstract
As part of the viral infection cycle, viruses must package their newly replicated genomes for delivery to other host cells. Bacteriophage φ29 packages its 6.6-µm long, double-stranded DNA into a 42 × 54 nm capsid1 by means of a portal complex that hydrolyses ATP2. This process is remarkable because entropic, electrostatic and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. Here we use optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force-generating motor. This motor can work against loads of up to 57 pN on average, making it one of the strongest molecular motors reported to date. Movements of over 5 µm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force–velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Notably, the packaging rate decreases as the prohead is filled, indicating that an internal force builds up to ∼50 pN owing to DNA confinement. Our data suggest that this force may be available for initiating the ejection of the DNA from the capsid during infection.
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References
Tao, Y. et al. Assembly of a tailed bacterial virus and its genome release studied in three dimensions. Cell 95, 431–437 (1998).
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).
Anderson, D. & Reilly, B. in Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics (eds Sonenshein, A., Hoch, J. A. & Losick, R.) 859–867 (American Society for Microbiology, Washington DC, 1993).
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).
Grimes, S. & Anderson, D. In vitro packaging of bacteriophage φ29 DNA restriction fragments and the role of the terminal protein gp3. J. Mol. Biol. 209, 91–100 (1989).
Simpson, A. A. et al. Structure of the bacteriophage φ29 DNA packaging motor. Nature 408, 745–750 (2000).
Ibarra, B. et al. Topology of the components of the DNA packaging machinery in the phage φ29 prohead. J. Mol. Biol. 298, 807–815 (2000).
Smith, S. B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).
Barlow, R. Statistics: a Guide to the Use of Statistical Methods in the Physical Sciences 15–16 (Wiley, Chichester, 1989).
Wang, M. D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1998).
Berg, H. C. & Turner, L. Torque generated by the flagellar motor of Escherichia coli. Biophys. J. 65, 2201–2216 (1993).
Kramers, H. Brownian motion in a field of force and the diffusion model of chemical reactions. Physica 7, 284–304 (1940).
Svoboda, K. & Block, S. M. Force and velocity measured for single kinesin molecules. Cell 77, 773–784 (1994).
Coppin, C. M., Pierce, D. W., Hsu, L. & Vale, R. D. The load dependence of kinesin's mechanical cycle. Proc. Natl Acad. Sci. USA 94, 8539–8544 (1997).
Finer, J. T., Simmons, R. M. & Spudich, J. A. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368, 113–119 (1994).
Lehninger, A. L., Nelson, D. L. & Cox, M. M. Principles of Biochemistry 375 (Worth, New York, 1993).
Earnshaw, W. C. & Casjens, S. R. DNA packaging by the double-stranded DNA bacteriophages. Cell 21, 319–331 (1980).
Rau, D. C., Lee, B. & Parsegian, V. A. Measurement of the repulsive force between polyelectrolyte molecules in ionic solution: hydration forces between parallel DNA double helices. Proc. Natl Acad. Sci. USA 81, 2621–2625 (1984).
Riemer, S. C. & Bloomfield, V. A. Packaging of DNA in bacteriophage heads: some considerations on energetics. Biopolymers 17, 785–794 (1978).
Odijk, T. Hexagonally packed DNA within bacteriophage T7 stabilized by curvature stress. Biophys. J. 75, 1223–1227 (1998).
Garcia, L. R. & Molineux, I. J. Transcription-independent DNA translocation of bacteriophage T7 DNA into Escherichia coli. J. Bacteriol. 178, 6921–6929 (1996).
Novick, S. L. & Baldeschwieler, J. D. Fluorescence measurement of the kinetics of DNA injection by bacteriophage lambda into liposomes. Biochemistry 27, 7919–7924 (1988).
Grimes, S. & Anderson, D. The bacteriophage φ29 packaging proteins supercoil the DNA ends. J. Mol. Biol. 266, 901–914 (1997).
Bjornsti, M. A., Reilly, B. E. & Anderson, D. L. Morphogenesis of bacteriophage φ29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA-gp3. J. Virol. 45, 383–396 (1983).
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).
Acknowledgements
We thank W. M. Gelbart, P. Jardine, T. Odijk, V. Bloomfield, D. Frenkel, C. Varga, A. Mehta and M. Young for comments. This research was supported in part by grants from the NIH, DOE and NSF. D.E.S. and S.J.T. are supported by a grant from the Packard Foundation. S.J.T. is supported by the Netherlands Organization for Scientific Research (NWO).
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Smith, D., Tans, S., Smith, S. et al. The bacteriophage φ29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001). https://doi.org/10.1038/35099581
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DOI: https://doi.org/10.1038/35099581
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