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Three-dimensional RNA structure refinement by hydroxyl radical probing

Nature Methods volume 9, pages 603–608 (2012)Cite this article

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Abstract

Molecular modeling guided by experimentally derived structural information is an attractive approach for three-dimensional structure determination of complex RNAs that are not amenable to study by high-resolution methods. Hydroxyl radical probing (HRP), which is performed routinely in many laboratories, provides a measure of solvent accessibility at individual nucleotides. HRP measurements have, to date, only been used to evaluate RNA models qualitatively. Here we report the development of a quantitative structure refinement approach using HRP measurements to drive discrete molecular dynamics simulations for RNAs ranging in size from 80 to 230 nucleotides. We first used HRP reactivities to identify RNAs that form extensive helical packing interactions. For these RNAs, we achieved highly significant structure predictions given the inputs of RNA sequence and base pairing. This HRP-directed tertiary structure refinement approach generates robust structural hypotheses that are useful for guiding explorations of structure-function inter-relationships in RNA.

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Figure 1: The relationship between RNA structure and HRP reactivity.
Figure 2: The assignment of potentials for incorporating HRP reactivities into DMD simulations.
Figure 3: HRP-directed RNA fold refinement for the training set.

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References

  1. Gesteland, R.F., Cech, T.R. & Atkins, J.F. eds. The RNA World: The Nature of Modern RNA Suggests a Prebiotic RNA World. (Cold Spring Harbor Lab Press, Plainview, NY, 2006).

  2. Das, R. & Baker, D. Automated de novo prediction of native-like RNA tertiary structures. Proc. Natl. Acad. Sci. USA 104, 14664–14669 (2007).

    Article  CAS  Google Scholar 

  3. Ding, F. et al. Ab initio RNA folding by discrete molecular dynamics: from structure prediction to folding mechanisms. RNA 14, 1164–1173 (2008).

    Article  CAS  Google Scholar 

  4. Parisien, M. & Major, F. The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature 452, 51–55 (2008).

    Article  CAS  Google Scholar 

  5. Cao, S. & Chen, S.J. Physics-based de novo prediction of RNA 3D structures. J. Phys. Chem. B 115, 4216–4226 (2011).

    Article  CAS  Google Scholar 

  6. Das, R. et al. Structural inference of native and partially folded RNA by high-throughput contact mapping. Proc. Natl. Acad. Sci. USA 105, 4144–4149 (2008).

    Article  CAS  Google Scholar 

  7. Yu, E.T., Hawkins, A., Eaton, J. & Fabris, D. MS3D structural elucidation of the HIV-1 packaging signal. Proc. Natl. Acad. Sci. USA 105, 12248–12253 (2008).

    Article  CAS  Google Scholar 

  8. Gherghe, C.M. et al. Native-like RNA tertiary structures using a sequence-encoded cleavage agent and refinement by discrete molecular dynamics. J. Am. Chem. Soc. 131, 2541–2546 (2009).

    Article  CAS  Google Scholar 

  9. Jonikas, M.A. et al. Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters. RNA 15, 189–199 (2009).

    Article  CAS  Google Scholar 

  10. Lavender, C.A., Ding, F., Dokholyan, N.V. & Weeks, K.M. Robust and generic RNA modeling using inferred constraints: a structure for the hepatitis C virus IRES pseudoknot domain. Biochemistry 49, 4931–4933 (2010).

    Article  CAS  Google Scholar 

  11. Yang, S., Parisien, M., Major, F. & Roux, B. RNA structure determination using SAXS data. J. Phys. Chem. B 114, 10039–10048 (2010).

    Article  CAS  Google Scholar 

  12. Michel, F. & Westhof, E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol. Biol. 216, 585–610 (1990).

    Article  CAS  Google Scholar 

  13. Gutell, R.R. et al. Identifying constraints on the higher-order structure of RNA: continued development and application of comparative sequence analysis methods. Nucleic Acids Res. 20, 5785–5795 (1992).

    Article  CAS  Google Scholar 

  14. Deigan, K.E., Li, T.W., Mathews, D.H. & Weeks, K.M. Accurate SHAPE-directed RNA structure determination. Proc. Natl. Acad. Sci. USA 106, 97–102 (2009).

    Article  CAS  Google Scholar 

  15. Weeks, K.M. Advances in RNA structure analysis by chemical probing. Curr. Opin. Struct. Biol. 20, 295–304 (2010).

    Article  CAS  Google Scholar 

  16. Hajdin, C.E., Ding, F., Dokholyan, N.V. & Weeks, K.M. On the significance of an RNA tertiary structure prediction. RNA 16, 1340–1349 (2010).

    Article  CAS  Google Scholar 

  17. Bailor, M.H., Mustoe, A.M., Brooks, C.L. III & Al-Hashimi, H.M. Topological constraints: using RNA secondary structure to model 3D conformation, folding pathways, and dynamic adaptation. Curr. Opin. Struct. Biol. 21, 296–305 (2011).

    Article  CAS  Google Scholar 

  18. Tullius, T.D. & Greenbaum, J.A. Mapping nucleic acid structure by hydroxyl radical cleavage. Curr. Opin. Chem. Biol. 9, 127–134 (2005).

    Article  CAS  Google Scholar 

  19. Cate, J.H. et al. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678–1685 (1996).

    Article  CAS  Google Scholar 

  20. Pastor, N., Weinstein, H., Jamison, E. & Brenowitz, M. A detailed interpretation of OH radical footprints in a TBP-DNA complex reveals the role of dynamics in the mechanism of sequence-specific binding. J. Mol. Biol. 304, 55–68 (2000).

    Article  CAS  Google Scholar 

  21. Bergman, N.H. et al. The three-dimensional architecture of the class I ligase ribozyme. RNA 10, 176–184 (2004).

    Article  CAS  Google Scholar 

  22. Rangan, P., Masquida, B., Westhof, E. & Woodson, S.A. Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme. Proc. Natl. Acad. Sci. USA 100, 1574–1579 (2003).

    Article  CAS  Google Scholar 

  23. Dokholyan, N.V., Buldyrev, S.V., Stanley, H.E. & Shakhnovich, E.I. Discrete molecular dynamics studies of the folding of a protein-like model. Fold. Des. 3, 577–587 (1998).

    Article  CAS  Google Scholar 

  24. Dann, C.E. III et al. Structure and mechanism of a metal-sensing regulatory RNA. Cell 130, 878–892 (2007).

    Article  CAS  Google Scholar 

  25. Balasubramanian, B., Pogozelski, W.K. & Tullius, T.D. DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc. Natl. Acad. Sci. USA 95, 9738–9743 (1998).

    Article  CAS  Google Scholar 

  26. Berman, H.M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

    Article  CAS  Google Scholar 

  27. Westhof, E., Dumas, P. & Moras, D. Restrained refinement of 2 crystalline forms of yeast aspartic-acid and phenylalanine transfer-RNA crystals. Acta Crystallogr. A 44, 112–123 (1988).

    Article  Google Scholar 

  28. Serganov, A. et al. Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch. Nature 441, 1167–1171 (2006).

    Article  CAS  Google Scholar 

  29. Krasilnikov, A.S., Yang, X., Pan, T. & Mondragon, A. Crystal structure of the specificity domain of ribonuclease P. Nature 421, 760–764 (2003).

    Article  CAS  Google Scholar 

  30. Adams, P.L. et al. Crystal structure of a self-splicing group I intron with both exons. Nature 430, 45–50 (2004).

    Article  CAS  Google Scholar 

  31. Cochrane, J.C., Lipchock, S.V., Smith, K.D. & Strobel, S.A. Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme. Biochemistry 48, 3239–3246 (2009).

    Article  CAS  Google Scholar 

  32. Serganov, A., Huang, L. & Patel, D.J. Structural insights into amino acid binding and gene control by a lysine riboswitch. Nature 455, 1263–1267 (2008).

    Article  CAS  Google Scholar 

  33. Kazantsev, A.V., Krivenko, A.A. & Pace, N.R. Mapping metal-binding sites in the catalytic domain of bacterial RNase P RNA. RNA 15, 266–276 (2009).

    Article  CAS  Google Scholar 

  34. Toor, N. et al. Tertiary architecture of the Oceanobacillus iheyensis group II intron. RNA 16, 57–69 (2010).

    Article  CAS  Google Scholar 

  35. Milligan, J.F., Groebe, D.R., Witherell, G.W. & Uhlenbeck, O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).

    Article  CAS  Google Scholar 

  36. Merino, E.J., Wilkinson, K.A., Coughlan, J.L. & Weeks, K.M. RNA structure analysis at single nucleotide resolution by Selective 2′-Hydroxyl Acylation and Primer Extension (SHAPE). J. Am. Chem. Soc. 127, 4223–4231 (2005).

    Article  CAS  Google Scholar 

  37. Duncan, C.D.S. & Weeks, K.M. The Mrs1 splicing factor binds the bI3 group I intron at each of two tetraloop-receptor motifs. PLoS One 5, e8983 (2010).

    Article  Google Scholar 

  38. Murphy, F.L. & Cech, T.R. An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme. Biochemistry 32, 5291–5300 (1993).

    Article  CAS  Google Scholar 

  39. Latham, J.A. & Cech, T.R. Defining the inside and outside of a catalytic RNA molecule. Science 245, 276–282 (1989).

    Article  CAS  Google Scholar 

  40. Klein, D.J., Been, M.D. & Ferre-D'Amare, A.R. Essential role of an active-site guanine in glmS ribozyme catalysis. J. Am. Chem. Soc. 129, 14858–14859 (2007).

    Article  CAS  Google Scholar 

  41. McGinnis, J.L., Duncan, C.D. & Weeks, K.M. High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly. Methods Enzymol. 468, 67–89 (2009).

    Article  CAS  Google Scholar 

  42. Vasa, S.M. et al. ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis. RNA 14, 1979–1990 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank E.A. Proctor, R. Redler and S. Ramachandran for critical readings of the manuscript. This work was supported by grants from the US National Institutes of Health to K.M.W. (GM064803) and N.V.D. (GM080742 and CA084480), by a US National Institutes of Health American Recovery and Reinvestment Act supplement (to K.M.W.) and by the University of North Carolina Research Council (to F.D.).

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA

    Feng Ding & Nikolay V Dokholyan

  2. Center for Computational and Systems Biology, University of North Carolina, Chapel Hill, North Carolina, USA

    Feng Ding & Nikolay V Dokholyan

  3. Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA

    Christopher A Lavender & Kevin M Weeks

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  1. Feng Ding
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  4. Nikolay V Dokholyan
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Contributions

F.D., K.M.W. and N.V.D. conceived of and designed the computational and experimental procedures. C.A.L. performed and analyzed the HRP measurements. F.D. developed the computational methodology and performed the computational analysis. F.D., C.A.L., K.M.W. and N.V.D. wrote the manuscript.

Corresponding authors

Correspondence to Kevin M Weeks or Nikolay V Dokholyan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2 (PDF 4812 kb)

Supplementary Dataset

The HRP reactivities and interaction parameters for all RNAs (XLS 284 kb)

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Ding, F., Lavender, C., Weeks, K. et al. Three-dimensional RNA structure refinement by hydroxyl radical probing. Nat Methods 9, 603–608 (2012). https://doi.org/10.1038/nmeth.1976

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