Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases

Nature Structural & Molecular Biology volume 13, pages 981–986 (2006)Cite this article

Abstract

Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Ded1 unwinds RNA duplexes irrespective of the orientation of single-stranded regions.
Figure 2: Supplementing single-stranded RNA in trans does not enhance the basal unwinding rate for the blunt-end substrates.
Figure 3: Equilibrium binding of different substrates by Ded1.
Figure 4: Unwinding of RNA-DNA hybrid substrates by Ded1.
Figure 5: Unwinding of MPS I by Ded1 but not by NPH-II.
Figure 6: Unwinding of MPS II by Ded1 but not by NPH-II, depicted as in Figure 5.
Figure 7: Unwinding of MPS III by Ded1 but not by NPH-II, with asterisk marking labeled RNA strand, gray circles symbolizing biotin and patterned shape symbolizing streptavidin.

Similar content being viewed by others

References

  1. Cordin, O., Banroques, J., Tanner, N.K. & Linder, P. The DEAD-box protein family of RNA helicases. Gene 367, 17–37 (2006).

    Article  CAS  Google Scholar 

  2. Eoff, R.L. & Raney, K.D. Helicase-catalysed translocation and strand separation. Biochem. Soc. Trans. 33, 1474–1478 (2005).

    Article  CAS  Google Scholar 

  3. Caruthers, J.M. & McKay, D.B. Helicase structure and mechanism. Curr. Opin. Struct. Biol. 12, 123–133 (2002).

    Article  CAS  Google Scholar 

  4. Tanner, N.K. & Linder, P. DExD/H box RNA helicases. From generic motors to specific dissociation functions. Mol. Cell 8, 251–261 (2001).

    Article  CAS  Google Scholar 

  5. Bowers, H.A. et al. Discriminatory RNP remodeling by the DEAD-box protein DED1. RNA 12, 903–912 (2006).

    Article  CAS  Google Scholar 

  6. Fairman, M.E. et al. Protein displacement by DExH/D RNA helicases without duplex unwinding. Science 304, 730–734 (2004).

    Article  CAS  Google Scholar 

  7. Linder, P. The life of RNA with proteins. Science 304, 694–695 (2004).

    Article  CAS  Google Scholar 

  8. Will, C.L. & Luehrmann, R. RNP remodeling with DExH/D boxes. Science 291, 1916–1917 (2001).

    Article  CAS  Google Scholar 

  9. Byrd, A.K. & Raney, K.D. Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA. Nat. Struct. Mol. Biol. 11, 531–538 (2004).

    Article  CAS  Google Scholar 

  10. Dumont, S. et al. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108 (2006).

    Article  CAS  Google Scholar 

  11. Jankowsky, E., Gross, C.H., Shuman, S. & Pyle, A.M. The DExH protein NPH-II is a processive and directional motor for unwinding RNA. Nature 403, 447–451 (2000).

    Article  CAS  Google Scholar 

  12. Myong, S., Rasnik, I., Joo, C., Lohman, T.M. & Ha, T. Repetitive shuttling of a motor protein on DNA. Nature 437, 1321–1325 (2005).

    Article  CAS  Google Scholar 

  13. Kawaoka, J., Jankowsky, E. & Pyle, A.M. Backbone tracking by the SF2 helicase NPH-II. Nat. Struct. Mol. Biol. 11, 526–530 (2004).

    Article  CAS  Google Scholar 

  14. von Hippel, P.H. Helicases become mechanistically simpler and functionally more complex. Nat. Struct. Mol. Biol. 11, 494–496 (2004).

    Article  CAS  Google Scholar 

  15. Lohman, T.M. & Bjornson, K.P. Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169–214 (1996).

    Article  CAS  Google Scholar 

  16. Jankowsky, E., Fairman, M.E. & Yang, Q. RNA helicases: versatile ATP-driven nanomotors. J. Nanosci. Nanotechnol. 5, 1983–1989 (2005).

    Article  CAS  Google Scholar 

  17. Rogers, G.W.J., Lima, W.F. & Merrick, W.C. Further characterization of the helicase activity of eIF4A. Substrate specificity. J. Biol. Chem. 276, 12598–12608 (2001).

    Article  CAS  Google Scholar 

  18. Huang, Y. & Liu, Z.R. The ATPase, RNA unwinding, and RNA binding activities of recombinant p68 RNA helicase. J. Biol. Chem. 277, 12810–12815 (2002).

    Article  CAS  Google Scholar 

  19. Bizebard, T., Ferlenghi, I., Iost, I. & Dreyfus, M. Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases. Biochemistry 43, 7857–7866 (2004).

    Article  CAS  Google Scholar 

  20. Linder, P. Yeast RNA helicases of the DEAD-box family involved in translation initiation. Biol. Cell 95, 157–167 (2003).

    Article  CAS  Google Scholar 

  21. Chuang, R.Y., Weaver, P.L., Liu, Z. & Chang, T.H. Requirement of the DEAD-Box protein ded1p for messenger RNA translation. Science 275, 1468–1471 (1997).

    Article  CAS  Google Scholar 

  22. Iost, I., Dreyfus, M. & Linder, P. Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase. J. Biol. Chem. 274, 17677–17683 (1999).

    Article  CAS  Google Scholar 

  23. Yang, Q. & Jankowsky, E. ATP- and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box protein DED1. Biochemistry 44, 13591–13601 (2005).

    Article  CAS  Google Scholar 

  24. Singleton, M.R., Dillingham, M.S., Gaudier, M., Kowalczykowski, S.C. & Wigley, D.B. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004).

    Article  CAS  Google Scholar 

  25. Dillingham, M.S., Spies, M. & Kowalczykowski, S.C. RecBCD enzyme is a bipolar DNA helicase. Nature 423, 893–897 (2003).

    Article  CAS  Google Scholar 

  26. Cordin, O., Tanner, N.K., Doere, M., Linder, P. & Banroques, J. The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity. EMBO J. 23, 2478–2487 (2004).

    Article  CAS  Google Scholar 

  27. Tanaka, N. & Schwer, B. Characterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22. Biochemistry 44, 9795–9803 (2005).

    Article  CAS  Google Scholar 

  28. Tanaka, N. & Schwer, B. Mutations in PRP43 that uncouple RNA-dependent NTPase activity and pre-mRNA splicing function. Biochemistry 45, 6510–6521 (2006).

    Article  CAS  Google Scholar 

  29. Kawaoka, J. & Pyle, A.M. Choosing between DNA and RNA: the polymer specificity of RNA helicase NPH-II. Nucleic Acids Res. 33, 644–649 (2005).

    Article  CAS  Google Scholar 

  30. Shuman, S. Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases. Proc. Natl. Acad. Sci. USA 89, 10935–10939 (1992).

    Article  CAS  Google Scholar 

  31. Shuman, S. Vaccinia virus RNA helicase. Directionality and substrate specificity. J. Biol. Chem. 268, 11798–11802 (1993).

    CAS  PubMed  Google Scholar 

  32. Beran, R.K., Bruno, M.M., Bowers, H.A., Jankowsky, E. & Pyle, A.M. Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track. J. Mol. Biol. 358, 974–982 (2006).

    Article  CAS  Google Scholar 

  33. Bianco, P.R. & Kowalczykowski, S.C. Translocation step size and mechanism of the RecBC DNA helicase. Nature 405, 368–372 (2000).

    Article  CAS  Google Scholar 

  34. Rocak, S. & Linder, P. DEAD-box proteins: the driving forces behind RNA metabolism. Nat. Rev. Mol. Cell Biol. 5, 232–241 (2004).

    Article  CAS  Google Scholar 

  35. Sengoku, T., Nureki, O., Nakamura, A., Kobayashi, S. & Yokoyama, S. Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa. Cell 125, 287–300 (2006).

    Article  CAS  Google Scholar 

  36. Bono, F., Ebert, J., Lorentzen, E. & Conti, E. The crystal structure of the exon junction complex reveals how it maintains a stable grip on mRNA. Cell 126, 713–725 (2006).

    Article  CAS  Google Scholar 

  37. Serebrov, V. & Pyle, A.M. Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430, 476–480 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Fairman for purification of NPH-II and A. Pyle, T. Nilsen, W. Merrick, J. Coller, M. Caprara and N. Kaye for comments on the manuscript. This work was supported by a grant from the US National Institutes of Health to E.J. E.J. is a Scholar of the Damon Runyon Cancer Research Foundation.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, 44106, Ohio, USA

    Quansheng Yang & Eckhard Jankowsky

Authors
  1. Quansheng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eckhard Jankowsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Q.Y. conducted experiments. Q.Y. and E.J. conceived and planned experiments, analyzed and interpreted data and wrote the manuscript.

Corresponding author

Correspondence to Eckhard Jankowsky.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Unwinding of MPS III components without streptavidin. (PDF 46 kb)

Supplementary Fig. 2

Unwinding reactions under single-turnover conditions. (PDF 169 kb)

Supplementary Fig. 3

Representative time courses for duplex unwinding. (PDF 43 kb)

Supplementary Fig. 4

Sequences of substrates used in Figures 1, 2, 3. (PDF 13 kb)

Supplementary Fig. 5

Unwinding of the 16-bp blunt-end RNA duplex requires ATP hydrolysis. (PDF 51 kb)

Supplementary Fig. 6

Sequences and functional characterization of substrates used in Figure 4. (PDF 67 kb)

Supplementary Fig. 7

Sequences of multipiece substrates used in Figures 5, 6, 7. (PDF 16 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, Q., Jankowsky, E. The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases. Nat Struct Mol Biol 13, 981–986 (2006). https://doi.org/10.1038/nsmb1165

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1165

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing