Letter | Published:

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase

Naturevolume 409pages113115 (2001) | Download Citation

Subjects

Abstract

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein1 and non-claret disjunctional protein (ncd)2 rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly3. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA4 supports the general picture of tracking along the DNA helix5. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Vale, R. D. & Toyoshima, Y. Y. Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia. Cell 52, 459–469 (1988).

  2. 2

    Walker, R. A., Salmon, E. D. & Endow, S. A. The Drosophila claret segregation protein is a minus-end directed motor molecule. Nature 347, 780–782 (1990).

  3. 3

    Sase, I., Miyata, H., Ishiwata, S. & Kinosita, K. Jr Axial rotation of sliding actin filaments revealed by single-fluorophore imaging. Proc. Natl Acad. Sci. USA 94, 5646– 5650 (1997).

  4. 4

    Wang, J. C. & Lynch, A. S. Transcription and DNA supercoiling. Curr. Opin. Genet. Dev. 3, 764– 768 (1993).

  5. 5

    Cook, P. R. The organization of replication and transcription. Science 284, 1790–1795 (1999).

  6. 6

    Schafer, D. A., Gelles, J., Sheetz, M. P. & Landick, R. Transcription by single molecules of RNA polymerase observed by light microscopy. Nature 352, 444–448 (1991).

  7. 7

    Wang, M. D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1999).

  8. 8

    Guthold, M. et al. Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase. Biophys. J. 77, 2284–2294 (1999).

  9. 9

    Davenport, R. J., Wuite, G. J. L., Landick, R. & Bustamante, C. Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase. Science 287, 2497– 2500 (2000).

  10. 10

    Kabata, H. et al. Visualization of single molecules of RNA polymerase sliding along DNA. Science 262, 1561– 1563 (1993).

  11. 11

    Harada, Y. et al. Single molecule imaging of RNA polymerase-DNA interactions in real time. Biophys. J. 76, 709– 715 (1999).

  12. 12

    Levin, J. R., Krummel, B. & Chamberlin, M. J. Isolation and properties of transcribing ternary complexes of Escherichia coli RNA polymerase positioned at single template base. J. Mol. Biol. 196, 85– 100 (1987).

  13. 13

    Stryer, L. Biochemistry 4th edn (Freeman, New York, 1995).

  14. 14

    Svoboda, K. & Block, S. M. Biological applications of optical tweezers. Annu. Rev. Biophys. Biomol. Struct. 23, 247–285 (1994).

  15. 15

    Strick, T., Allemand, J.-F., Bensimon, D., Lavery, R. & Croquette, V. Phase coexistence in a single DNA molecule. Physica A 263, 392– 405 (1999).

  16. 16

    Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. Jr Direct observation of the rotation of F1-ATPase. Nature 386, 299–302 ( 1997).

  17. 17

    Yasuda, R., Noji, H., Kinosita, K. Jr & Yoshida, M. F1-ATPase is a highly efficient molecular motor that rotates with discrete 120° steps. Cell 93, 1117– 1124 (1998).

  18. 18

    DeRosier, D. J. The turn of the screw: the bacterial flagellar motor. Cell 93, 17–20 (1998).

  19. 19

    Ryu, W. S., Berry, R. M. & Berg, H. C. Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio. Nature 403, 444–447 (2000).

  20. 20

    Adachi, K. et al. Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging. Proc. Natl Acad. Sci. USA 97, 7243–7247 ( 2000).

  21. 21

    Kubori, T. & Shimamoto, N. A branched pathway in the early stage of transcription by Escherichia coli RNA polymerase. J. Mol. Biol. 256, 449–457 (1996).

  22. 22

    Studier, F. W. Gene 0·3 of bacteriophage T7 acts to overcome the DNA restriction system of the host. J. Mol. Biol. 94, 283–295 (1975).

Download references

Acknowledgements

We thank M. Susa for help in transcription analysis; A. Ishihama, S. Ishiwata, G. W. Feigenson and members of Team 13 for comments; and H. Umezawa for laboratory management. This work was supported in part by Grants-in-Aid from Ministry of Education, Science, Sports and Culture of Japan, Hayashi Memorial Foundation for Female Natural Scientists, and an Academic Frontier Promotional Project.

Author information

Author notes

    • Yoshie Harada

    Present address: Department of Molecular Physiology, The Tokyo Metropolitan Institute of Medical Science, 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo, 113-8613, Japan

Affiliations

  1. Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, 223-8522, Yokohama, Japan

    • Yoshie Harada
    • , Akira Takatsuki
    •  & Kazuhiko Kinosita Jr
  2. CREST (Core Research for Evolutional Science and Technology) “Genetic Programming” Team 13, Nogawa 907, Miyamae-ku, 216-0001 , Kawasaki, Japan

    • Yoshie Harada
    • , Hiroyasu Itoh
    •  & Kazuhiko Kinosita Jr
  3. Kazusa DNA Research Institute, Yana 1532-3, Kisarazu, 292-0812, Japan

    • Osamu Ohara
  4. Tsukuba Research Laboratory, Hamamatsu Photonics KK, Tokodai, 300-2635, Tsukuba, Japan

    • Hiroyasu Itoh
  5. Structural Biology Center, National Institute of Genetics, Mishima, 411-8540, Japan

    • Nobuo Shimamoto

Authors

  1. Search for Yoshie Harada in:

  2. Search for Osamu Ohara in:

  3. Search for Akira Takatsuki in:

  4. Search for Hiroyasu Itoh in:

  5. Search for Nobuo Shimamoto in:

  6. Search for Kazuhiko Kinosita Jr in:

Corresponding author

Correspondence to Yoshie Harada.

Supplementary information

About this article

Publication history

Received

Accepted

Issue Date

DOI

https://doi.org/10.1038/35051126

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.