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Duplex Unwinding with DEAD-Box Proteins

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Helicases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 587))

Abstract

DEAD-box proteins, which comprise the largest helicase family, are involved in virtually all aspects of RNA metabolism. DEAD-box proteins catalyze diverse ATP-driven functions including the unwinding of RNA secondary structures. In contrast to many well-studied DNA and viral RNA helicases, DEAD-box proteins do not rely on translocation on one of the nucleic acid strands for duplex unwinding, but directly load onto helical regions and then locally pry the strands apart in an ATP-dependent fashion. In this chapter, we outline substrate design and unwinding protocols for DEAD-box proteins and focus on the quantitative evaluation of their unwinding activity.

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References

  1. Linder P. (2006) Dead-box proteins: a family affair—active and passive players in RNP-remodeling. Nucleic Acids Res. 34, 4168–4180.

    Article  PubMed  CAS  Google Scholar 

  2. Jankowsky E. and Fairman M. (2007) RNA helicases—one fold for many functions. Curr. Opin. Struct. Biol. 17, 316–324.

    Article  PubMed  CAS  Google Scholar 

  3. Zhang M. and Green M. R. (2001) Identification and characterization of yUAP/Sub2p, a yeast homolog of the essential human pre-mRNA splicing factor hUAP56. Genes Dev. 15, 30–35.

    Article  PubMed  Google Scholar 

  4. Linder P., Lasko P. F., Ashburner M., Leroy P., Nielsen P. J., Nishi K., Schnier J., and Slonimski P. P. (1989) Birth of the D-E-A-D box. Nature 337, 121–122.

    Article  PubMed  CAS  Google Scholar 

  5. Liang X. H. and Fournier M. J. (2006) The helicase Has1p is required for snoRNA release from pre-rRNA. Mol. Cell Biol. 26, 7437–7450.

    Article  PubMed  CAS  Google Scholar 

  6. Kos M. and Tollervey D. (2005) The putative RNA helicase Dbp4p is required for release of the U14 snoRNA from preribosomes in Saccharomces cerevisiae. Mol. Cell 20, 53–64.

    Article  PubMed  CAS  Google Scholar 

  7. Shibuya T., Tange T. O., Sonenberg N., and Moore M. J. (2004) eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 11, 346–351.

    Article  PubMed  CAS  Google Scholar 

  8. Ballut L., Marchadier B., Baguet A., Tomasetto C., Seraphin B., and Le Hir H. (2005) The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity. Nat. Struct. Mol. Biol. 12, 861–869.

    Article  PubMed  CAS  Google Scholar 

  9. Andersen C. B., Ballut L., Johansen J. S., Chamieh H., Nielsen K. H., Oliveira C. L., Pedersen J. S., Seraphin B., Le Hir H., and Andersen G. R. (2006) Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA. Science 313, 1968–1972.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  11. Bowers H. A., Maroney P. A., Fairman M. E., Kastner B., Luhrmann R., Nilsen T. W., and Jankowsky E. (2006) Discriminatory RNP remodeling by the DEAD-box protein DED1. RNA 12, 903–912.

    Article  PubMed  CAS  Google Scholar 

  12. Fairman M., Maroney P. A., Wang W., Bowers H., Gollnick P., Nilsen T. W., and Jankowsky E. (2004) Protein displacement by DExH/D RNA helicases without duplex unwinding. Science 304, 730–734.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  14. Jankowsky E. and Fairman M. (2008) in RNA-Protein Interaction Protocols, Ed. Lin R. J. Humana Press, Totowa, NJ, Vol. 488, pp. 343–355.

    Chapter  Google Scholar 

  15. Tijerina P., Bhaskaran H., and Russell R. (2006) Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone. Proc. Natl. Acad. Sci. U.S.A. 103, 16698–16703.

    Article  PubMed  CAS  Google Scholar 

  16. Yang Q. and Jankowsky E. (2006) The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases. Nat. Struct. Mol. Biol. 13, 981–986.

    Article  PubMed  CAS  Google Scholar 

  17. Yang Q., Del Campo M., Lambowitz A. M., and Jankowsky E. (2007) DEAD-box proteins unwind duplexes by local strand separation. Mol. Cell 28, 253–263.

    Article  PubMed  CAS  Google Scholar 

  18. 18.Liu F., Putnam A., and Jankowsky E. (2008) ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding. Proc. Natl. Acad. Sci. U.S.A. 105, 20209–20214.

    Article  PubMed  CAS  Google Scholar 

  19. Chen Y., and Russell R. (2008) The DEAD-box protein CYT-19 uses a single ATP to completely separate a short RNA duplex. Proc. Natl. Acad. Sci. U.S.A. 105, 20203–20209.

    Article  PubMed  CAS  Google Scholar 

  20. Rogers G. W., Richter N. J., and Merrick W. C. (1999) Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A. J. Biol. Chem. 274, 12236–12244.

    Article  PubMed  CAS  Google Scholar 

  21. Del Campo M., Tijerina P., Bhaskaran H., Mohr S., Yang Q., Jankowsky E., Russell R., and Lambowitz A. M. (2007) Do DEAD-box proteins promote group II intron splicing without unwinding RNA? Mol. Cell 28, 159–166.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  24. Diges C. M. and Uhlenbeck O. C. (2001) Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA. EMBO J. 20, 5503–5512.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  26. Rogers G. W. J., Richter N. J., and Lima WF, M. W. (2001) Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F. J. Biol. Chem. 276, 30914–30922.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  28. Yang Q., Fairman M. E., and Jankowsky E. (2007) DEAD-box-protein-assisted RNA structure conversion towards and against thermodynamic equilibrium values. J. Mol. Biol. 368, 1087–1100.

    Article  PubMed  CAS  Google Scholar 

  29. Chamot D., Colvin K. R., Kujat-Choy S. L., and Owttrim G. W. (2005) RNA structural rearrangement via unwinding and annealing by the cyanobacterial RNA helicase CrhR. J. Biol. Chem. 280, 2036–2044.

    Article  PubMed  CAS  Google Scholar 

  30. Rossler O. G., Straka A., and Stahl H. (2001) Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72. Nucleic Acids Res. 29, 2088–2096.

    Article  PubMed  CAS  Google Scholar 

  31. Ali J. A. and Lohman T. M. (1997) Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 275, 377–380.

    Article  PubMed  CAS  Google Scholar 

  32. Lorsch J. R. and Herschlag D. (1998) The DEAD box protein eIF4A. 1. A minimal kinetic and thermodynamic framework reveals coupled binding of RNA and nucleotide. Biochemistry 37, 2180–2193.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Eckhard Jankowsky .

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Jankowsky, E., Putnam, A. (2009). Duplex Unwinding with DEAD-Box Proteins. In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_18

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  • DOI: https://doi.org/10.1007/978-1-60327-355-8_18

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-354-1

  • Online ISBN: 978-1-60327-355-8

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