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Crystal structure of group II intron domain 1 reveals a template for RNA assembly

Nature Chemical Biology volume 11, pages 967–972 (2015)Cite this article

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Abstract

Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. Here, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Composed of domain 1 from the Oceanobacillus iheyensis group II intron (266 nucleotides), this intermediate retains native-like features but adopts a compact conformation in which the active site cleft is closed. Transition between this closed and the open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. The open state is then stabilized by sequential docking of downstream intron domains, suggesting a 'first come, first folded' strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures.

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Figure 1: Structure of D1 in the presence and absence of downstream domains.
Figure 2: Crystallographic B-factor distribution in D1iso.
Figure 3: Box plot showing difference average B-factors between the junction residues (denoted as ΔBjunction on y-axis) and specific motifs taken from D1iso, D1full and from all RNA entries in PDB (x-axis).
Figure 4: Difference pseudo-torsion angle (Δ(η,θ)) between D1iso and D1full.
Figure 5: Group II intron folding assembly pathway.

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References

  1. Treiber, D.K. & Williamson, J.R. Beyond kinetic traps in RNA folding. Curr. Opin. Struct. Biol. 11, 309–314 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Woodson, S.A. Recent insights on RNA folding mechanisms from catalytic RNA. Cell. Mol. Life Sci. 57, 796–808 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Sosnick, T.R. & Pan, T. RNA folding: models and perspectives. Curr. Opin. Struct. Biol. 13, 309–316 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Su, L.J., Waldsich, C. & Pyle, A.M. An obligate intermediate along the slow folding pathway of a group II intron ribozyme. Nucleic Acids Res. 33, 6674–6687 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Woodson, S.A. Compact intermediates in RNA folding. Annu. Rev. Biophys. 39, 61–77 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bryngelson, J.D., Onuchic, J.N., Socci, N.D. & Wolynes, P.G. Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins 21, 167–195 (1995).

    Article  CAS  PubMed  Google Scholar 

  7. Kim, P.S. & Baldwin, R.L. Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660 (1990).

    Article  CAS  PubMed  Google Scholar 

  8. Dyson, H.J. & Wright, P.E. Peptide conformation and protein-folding. Curr. Opin. Struct. Biol. 3, 60–65 (1993).

    Article  CAS  Google Scholar 

  9. Pyle, A.M. The tertiary structure of group II introns: implications for biological function and evolution. Crit. Rev. Biochem. Mol. Biol. 45, 215–232 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pyle, A.M. & Lambowitz, A.M. Group II introns: ribozymes that splice RNA and invade DNA. in The RNA World 3rd edn. (ed. Gesteland, R.F.) 469–505 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2006).

  11. Pyle, A.M., Fedorova, O. & Waldsich, C. Folding of group II introns: a model system for large, multidomain RNAs? Trends Biochem. Sci. 32, 138–145 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Krasilnikov, A.S., Xiao, Y., Pan, T. & Mondragon, A. Basis for structural diversity in homologous RNAs. Science 306, 104–107 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Marcia, M. & Pyle, A.M. Visualizing group II intron catalysis through the stages of splicing. Cell 151, 497–507 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Toor, N., Keating, K.S., Taylor, S.D. & Pyle, A.M. Crystal structure of a self-spliced group II intron. Science 320, 77–82 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Coonrod, L.A., Lohman, J.R. & Berglund, J.A. Utilizing the GAAA tetraloop/receptor to facilitate crystal packing and determination of the structure of a CUG RNA helix. Biochemistry 51, 8330–8337 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Marcia, M. et al. Solving nucleic acid structures by molecular replacement: examples from group II intron studies. Acta Crystallogr. D Biol. Crystallogr. 69, 2174–2185 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Robertson, M.P. & Scott, W.G. A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives. Acta Crystallogr. D Biol. Crystallogr. D64, 738–744 (2008).

    Article  PubMed  CAS  Google Scholar 

  19. Marcia, M. & Pyle, A.M. Principles of ion recognition in RNA: insights from the group II intron structures. RNA 20, 516–527 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Qin, P.Z. & Pyle, A.M. Stopped-flow fluorescence spectroscopy of a group II intron ribozyme reveals that domain 1 is an independent folding unit with a requirement for specific Mg2+ ions in the tertiary structure. Biochemistry 36, 4718–4730 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Michels, W.J. Jr. & Pyle, A.M. Conversion of a group II intron into a new multiple-turnover ribozyme that selectively cleaves oligonucleotides: elucidation of reaction mechanism and structure/function relationships. Biochemistry 34, 2965–2977 (1995).

    Article  CAS  PubMed  Google Scholar 

  22. Drenth, J. Theory of X-ray diffraction by a crystal. Principles of Protein X-Ray Crystallography Chapter 4, 64–108 (Springer, 2007).

  23. Mohan, S., Donohue, J.P. & Noller, H.F. Molecular mechanics of 30S subunit head rotation. Proc. Natl. Acad. Sci. USA 111, 13325–13330 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Keating, K.S., Humphris, E.L. & Pyle, A.M. A new way to see RNA. Q. Rev. Biophys. 44, 433–466 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Duarte, C.M., Wadley, L.M. & Pyle, A.M. RNA structure comparison, motif search and discovery using a reduced representation of RNA conformational space. Nucleic Acids Res. 31, 4755–4761 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Duarte, C.M. & Pyle, A.M. Stepping through an RNA structure: a novel approach to conformational analysis. J. Mol. Biol. 284, 1465–1478 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. Fedorova, O. & Pyle, A.M. The brace for a growing scaffold: Mss116 protein promotes RNA folding by stabilizing an early assembly intermediate. J. Mol. Biol. 422, 347–365 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Guo, F., Gooding, A.R. & Cech, T.R. Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site. Mol. Cell 16, 351–362 (2004).

    CAS  PubMed  Google Scholar 

  30. Juneau, K., Podell, E., Harrington, D.J. & Cech, T.R. Structural basis of the enhanced stability of a mutant ribozyme domain and a detailed view of RNA–solvent interactions. Structure 9, 221–231 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Waldsich, C. & Pyle, A.M. A kinetic intermediate that regulates proper folding of a group II intron RNA. J. Mol. Biol. 375, 572–580 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Konforti, B.B., Liu, Q. & Pyle, A.M. A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme. EMBO J. 17, 7105–7117 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Torres-Larios, A., Swinger, K.K., Krasilnikov, A.S., Pan, T. & Mondragon, A. Crystal structure of the RNA component of bacterial ribonuclease P. Nature 437, 584–587 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Baird, N.J., Westhof, E., Qin, H., Pan, T. & Sosnick, T.R. Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding. J. Mol. Biol. 352, 712–722 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Pan, T. Folding of bacterial RNase P RNA. in Protein Reviews Vol. Ribonuclease P (eds. Liu, F. & Altman, S.) 79–91 (Springer, 2010).

  36. 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  PubMed  PubMed Central  Google Scholar 

  37. Bailor, M.H., Sun, X. & Al-Hashimi, H.M. Topology links RNA secondary structure with global conformation, dynamics, and adaptation. Science 327, 202–206 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Chu, V.B. et al. Do conformational biases of simple helical junctions influence RNA folding stability and specificity? RNA 15, 2195–2205 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Behrouzi, R., Roh, J.H., Kilburn, D., Briber, R.M. & Woodson, S.A. Cooperative tertiary interaction network guides RNA folding. Cell 149, 348–357 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Johnson, T.H., Tijerina, P., Chadee, A.B., Herschlag, D. & Russell, R. Structural specificity conferred by a group I RNA peripheral element. Proc. Natl. Acad. Sci. USA 102, 10176–10181 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Pan, T. & Sosnick, T. RNA folding during transcription. Annu. Rev. Biophys. Biomol. Struct. 35, 161–175 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Somarowthu, S. et al. HOTAIR forms an intricate and modular secondary structure. Mol. Cell 58, 353–361 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Chillon, I. et al. Native purification and analysis of long RNAs. Methods Enzymol. 558, 3–37 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hughson, F.M., Wright, P.E. & Baldwin, R.L. Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544–1548 (1990).

    Article  CAS  PubMed  Google Scholar 

  45. Bhutani, N. & Udgaonkar, J.B. Folding subdomains of thioredoxin characterized by native-state hydrogen exchange. Protein Sci. 12, 1719–1731 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Llinás, M. & Marqusee, S. Subdomain interactions as a determinant in the folding and stability of T4 lysozyme. Protein Sci. 7, 96–104 (1998).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Wu, L.C., Grandori, R. & Carey, J. Autonomous subdomains in protein folding. Protein Sci. 3, 369–371 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Uzawa, T. et al. Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR. Proc. Natl. Acad. Sci. USA 105, 13859–13864 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chworos, A. et al. Building programmable jigsaw puzzles with RNA. Science 306, 2068–2072 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Han, D. et al. DNA origami with complex curvatures in three-dimensional space. Science 332, 342–346 (2011).

    Article  CAS  PubMed  Google Scholar 

  51. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Sheldrick, G.M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D Biol. Crystallogr. 66, 479–485 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  56. Keating, K.S. & Pyle, A.M. RCrane: semi-automated RNA model building. Acta Crystallogr. D Biol. Crystallogr. 68, 985–995 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Karplus, P.A. & Diederichs, K. Assessing and maximizing data quality in macromolecular crystallography. Curr. Opin. Struct. Biol. 34, 60–68 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Krebs, W.G. & Gerstein, M. The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework. Nucleic Acids Res. 28, 1665–1675 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Lu, X.J. & Olson, W.K. 3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures. Nucleic Acids Res. 31, 5108–5121 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Schneider, B., Gelly, J.C., de Brevern, A.G. & Cerny, J. Local dynamics of proteins and DNA evaluated from crystallographic B factors. Acta Crystallogr. D Biol. Crystallogr. 70, 2413–2419 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We would like to thank S. Somarowthu for constructive discussions and O. Fedorova, S. Somarowthu and T. Dickey for reading the manuscript. C.Z. is supported by Gruber Science Fellowship. A.M.P. is a Howard Hughes Medical Institute Investigator. This work is supported by the US National Institute of Health (NIH) (RO1GM50313) and is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (P41 GM103403). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

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  1. Marco Marcia

    Present address: Present address: European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 09, France.,

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Chen Zhao

  2. Northeastern Collaborative Access Team (NE-CAT), Argonne National Laboratory, Argonne, Illinois, USA

    Kanagalaghatta R Rajashankar

  3. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA

    Kanagalaghatta R Rajashankar

  4. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Marco Marcia & Anna Marie Pyle

  5. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Anna Marie Pyle

  6. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Anna Marie Pyle

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Contributions

C.Z. designed the constructs and conducted all the experiments; C.Z., K.R.R. and M.M. collected X-ray diffraction data; and C.Z., K.R.R. and M.M. solved the structure. C.Z. analyzed the structure. A.M.P. and M.M. directed the research. A.M.P. and C.Z. wrote the manuscript, and M.M. and K.R.R. contributed to writing.

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Correspondence to Marco Marcia or Anna Marie Pyle.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–8. (PDF 0 kb)

Morphing between D1iso and D1full and subsequent docking of D2–D5.

This movie starts with a 360° rotation of the D1iso. Subsequently, D1iso opens up to D1full, then D1full closes to D1iso. This process is repeated one more time with hinge residues located at 5′H and 3′H colored in green and pink. Finally, D1iso opens to D1full for one more time, then downstream domains D2–D5 dock sequentially. D1–D5 are colored in grey, blue, wheat, yellow and red respectively. The procedure used to produce this video is described in Online Methods section of the article. (MOV 24403 kb)

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Zhao, C., Rajashankar, K., Marcia, M. et al. Crystal structure of group II intron domain 1 reveals a template for RNA assembly. Nat Chem Biol 11, 967–972 (2015). https://doi.org/10.1038/nchembio.1949

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