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Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p

Nature volume 490, pages 121–125 (2012)Cite this article

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Abstract

DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life1,2. They contain a conserved ‘helicase core’ of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation3. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA–protein complexes without globally disrupting RNA structure4. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a ‘closed state’ with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

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Figure 1: The distinct substrate-binding characteristics of the helicase core domains of Mss116p.
Figure 2: Crystal structures of Mss116p D2 bound to A-form duplexes.
Figure 3: Interactions between Mss116p D2 and duplex RNA.
Figure 4: RNA-duplex binding and unwinding by Mss116p.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors were deposited in the Protein Data Bank under accessions 4DB2 (D2–dsRNA) and 4DB4 (D2–dsRNA–DNA).

References

  1. Linder, P. & Jankowsky, E. From unwinding to clamping—the DEAD box RNA helicase family. Nature Rev. Mol. Cell Biol. 12, 505–516 (2011)

    Article  CAS  Google Scholar 

  2. Jarmoskaite, I. & Russell, R. DEAD-box proteins as RNA helicases and chaperones. WIREs: RNA 2, 135–152 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Pan, C. & Russell, R. Roles of DEAD-box proteins in RNA and RNP folding. RNA Biol. 7, 667–676 (2010)

    Article  CAS  Google Scholar 

  5. Huang, H. R. et al. The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function. Proc. Natl Acad. Sci. USA 102, 163–168 (2005)

    Article  ADS  CAS  Google Scholar 

  6. Del Campo, M. et al. Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones. J. Mol. Biol. 389, 674–693 (2009)

    Article  CAS  Google Scholar 

  7. Potratz, J. P., Del Campo, M., Wolf, R. Z., Lambowitz, A. M. & Russell, R. ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo. J. Mol. Biol. 411, 661–679 (2011)

    Article  CAS  Google Scholar 

  8. Cao, W. et al. Mechanism of Mss116 ATPase reveals functional diversity of DEAD-box proteins. J. Mol. Biol. 409, 399–414 (2011)

    Article  CAS  Google Scholar 

  9. Mohr, G. et al. High-throughput genetic identification of functionally important regions of the yeast DEAD-box protein Mss116p. J. Mol. Biol. 413, 952–972 (2011)

    Article  CAS  Google Scholar 

  10. Del Campo, M. & Lambowitz, A. M. Structure of the yeast DEAD-box protein Mss116p reveals two wedges that crimp RNA. Mol. Cell 35, 598–609 (2009)

    Article  CAS  Google Scholar 

  11. Mallam, A. L. et al. Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail. Proc. Natl Acad. Sci. USA 108, 12254–12259 (2011)

    Article  ADS  CAS  Google Scholar 

  12. Wang, S., Overgaard, M. T., Hu, Y. & McKay, D. B. The Bacillus subtilis RNA helicase YxiN is distended in solution. Biophys. J. 94, L01–L03 (2008)

    Article  CAS  Google Scholar 

  13. 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 

  14. Rudolph, M. G., Heissmann, R., Wittmann, J. G. & Klostermeier, D. Crystal structure and nucleotide binding of the Thermus thermophilus RNA helicase Hera N-terminal domain. J. Mol. Biol. 361, 731–743 (2006)

    Article  CAS  Google Scholar 

  15. Schütz, P. et al. Comparative structural analysis of human DEAD-box RNA helicases. PLoS ONE 5, e12791 (2010)

    Article  ADS  Google Scholar 

  16. Egli, M., Usman, N. & Rich, A. Conformational influence of the ribose 2′-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes. Biochemistry 32, 3221–3237 (1993)

    Article  CAS  Google Scholar 

  17. Wahl, M. C. & Sundaralingam, M. B-form to A-form conversion by a 3′-terminal ribose: crystal structure of the chimera d(CCACTAGTG)r(G). Nucleic Acids Res. 28, 4356–4363 (2000)

    Article  CAS  Google Scholar 

  18. Halls, C. et al. Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity. J. Mol. Biol. 365, 835–855 (2007)

    Article  CAS  Google Scholar 

  19. Henn, A. et al. Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA. Proc. Natl Acad. Sci. USA 107, 4046–4050 (2010)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Chen, Y. et al. DEAD-box proteins can completely separate an RNA duplex using a single ATP. Proc. Natl Acad. Sci. USA 105, 20203–20208 (2008)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  23. Andersen, C. B. F. et al. Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA. Science 313, 1968–1972 (2006)

    Article  ADS  CAS  Google Scholar 

  24. Fairman-Williams, M. E., Guenther, U. P. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010)

    Article  CAS  Google Scholar 

  25. Singleton, M. R., Dillingham, M. S. & Wigley, D. B. Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50 (2007)

    Article  CAS  Google Scholar 

  26. Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Blinov, V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17, 4713–4730 (1989)

    Article  CAS  Google Scholar 

  27. Kowalinski, E. et al. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell 147, 423–435 (2011)

    Article  CAS  Google Scholar 

  28. Jiang, F. et al. Structural basis of RNA recognition and activation by innate immune receptor RIG-I. Nature 479, 423–427 (2011)

    Article  ADS  CAS  Google Scholar 

  29. Luo, D. et al. Structural insights into RNA recognition by RIG-I. Cell 147, 409–422 (2011)

    Article  CAS  Google Scholar 

  30. Ozalp, V. C., Pedersen, T. R., Nielsen, L. J. & Olsen, L. F. Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor. J. Biol. Chem. 285, 37579–37588 (2010)

    Article  Google Scholar 

  31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  32. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D 67, 293–302 (2011)

    Article  CAS  Google Scholar 

  33. Pflugrath, J. W. The finer things in X-ray diffraction data collection. Acta Crystallogr. D 55, 1718–1725 (1999)

    Article  CAS  Google Scholar 

  34. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  35. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  36. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  37. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  38. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

  39. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

    Article  CAS  Google Scholar 

  40. Wilkins, M. R. et al. Protein identification and analysis tools in the ExPASy server. Methods Mol. Biol. 112, 531–552 (1999)

    CAS  PubMed  Google Scholar 

  41. Tang, G. Q., Bandwar, R. P. & Patel, S. S. Extended upstream A-T sequence increases T7 promoter strength. J. Biol. Chem. 280, 40707–40713 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Monzingo (University of Texas at Austin Macromolecular Crystallography Facility) for help with X-ray diffraction data collection, and R. Russell (University of Texas at Austin) and E. Jankowsky (Case Western Reserve University) for comments on the manuscript. X-ray diffraction data were collected at the Berkeley Center for Structural Biology, which is supported in part by the National Institutes of Health (NIH), National Institute of General Medical Sciences and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract no. DE-AC02-05CH11231. A.L.M. is the recipient of an EMBO long-term fellowship (ALTF 389-2010). This work was supported by NIH grant GM037951.

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  1. Mark Del Campo

    Present address: Present address: Rigaku Americas Corporation, 9009 New Trails Drive, The Woodlands, Texas 77381, USA.,

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, 78712, Texas, USA

    Anna L. Mallam, Mark Del Campo, Benjamin Gilman, David J. Sidote & Alan M. Lambowitz

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Contributions

This study was designed by A.L.M. and A.M.L. A.L.M. cloned the individual domain constructs, purified proteins, crystallized the complexes, collected X-ray crystallographic data, and performed binding assays. A.L.M., M.D.C. and D.J.S. processed and refined the X-ray diffraction data. B.G. performed genetic assays. All authors contributed to analysing the results. A.L.M., M.D.C. and A.M.L. wrote the paper, with contributions from D.J.S. and B.G.

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Correspondence to Alan M. Lambowitz.

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The authors declare no competing financial interests.

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Mallam, A., Del Campo, M., Gilman, B. et al. Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p. Nature 490, 121–125 (2012). https://doi.org/10.1038/nature11402

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