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Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif

Abstract

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5γ in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg2+ binding to a minor groove shelf in the D5 bulge. Another distinct metal ion–binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.

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Figure 1: NMR structure of D5.
Figure 2: Solution structure of D5.
Figure 3: Change of chemical shifts in D5 upon Mg2+ binding.

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References

  1. Bonen, L. & Vogel, J. The ins and outs of group II introns. Trends Genet. 17, 322–331 (2001).

    Article  CAS  Google Scholar 

  2. Michel, F. & Ferat, J.L. Structure and activities of group II introns. Annu. Rev. Biochem. 64, 435–461 (1995).

    Article  CAS  Google Scholar 

  3. Sontheimer, E.J., Gordon, P.M. & Piccirilli, J.A. Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome. Genes Dev. 13, 1729–1741 (1999).

    Article  CAS  Google Scholar 

  4. Abramovitz, D.L., Friedman, R.A. & Pyle, A.M. Catalytic role of 2′-hydroxyl groups within a group II intron active site. Science 271, 1410–1413 (1996).

    Article  CAS  Google Scholar 

  5. Konforti, B.B. et al. Ribozyme catalysis from the major groove of group II intron domain 5. Mol. Cell 1, 433–441 (1998).

    Article  CAS  Google Scholar 

  6. Chanfreau, G. & Jacquier, A. Catalytic site components common to both splicing steps of a group II intron. Science 266, 1383–1387 (1994).

    Article  CAS  Google Scholar 

  7. Gordon, P.M. & Piccirilli, J.A. Metal ion coordination by the AGC triad in domain 5 contributes to group II intron catalysis. Nat. Struct. Biol. 8, 893–898 (2001).

    Article  CAS  Google Scholar 

  8. Boudvillain, M., deLencastre, A. & Pyle, A.M. A tertiary interaction that links active-site domains to the 5′ splice site of a group II intron. Nature 406, 315–318 (2000).

    Article  CAS  Google Scholar 

  9. Zhang, L. & Doudna, J.A. Structural insights into group II intron catalysis and branch-site selection. Science 295, 2084–2088 (2002).

    Article  CAS  Google Scholar 

  10. Costa, M., Michel, F. & Westhof, E. A three dimensional perspective on exon binding by a group II self-splicing intron. EMBO J. 19, 5007–5018 (2000).

    Article  CAS  Google Scholar 

  11. Swisher, J., Duarte, C.M., Su, L.J. & Pyle, A.M. Visualizing the solvent-inaccessible core of a group II intron ribozyme. EMBO J. 20, 2051–2061 (2001).

    Article  CAS  Google Scholar 

  12. Michel, F., Umesono, K. & Ozeki, H. Comparative and functional anatomy of group II catalytic introns—a review. Gene 82, 5–30 (1989).

    Article  CAS  Google Scholar 

  13. Costa, M., Christian, E.L. & Michel, F. Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V. RNA 4, 1055–1068 (1998).

    Article  CAS  Google Scholar 

  14. Sigel, R.K.O., Vaidya, A. & Pyle, A.M. Metal ion binding sites in a group II intron core. Nat. Struct. Biol. 7, 1111–1116 (2000).

    Article  CAS  Google Scholar 

  15. Hennig, M. & Williamson, J.R. Detection of N-H-N hydrogen bonding in RNA via scalar couplings in the absence of observable imino proton resonances. Nucleic Acids Res. 28, 1585–1593 (2000).

    Article  CAS  Google Scholar 

  16. Luy, B. & Marino, J.P. Direct evidence for Watson-Crick base pairs in a dynamic region of RNA structure. J. Am. Chem. Soc. 122, 8095–8096 (2000).

    Article  CAS  Google Scholar 

  17. Kolk, M.H. et al. NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA. Science 280, 434–438 (1998).

    Article  CAS  Google Scholar 

  18. Heus, H.A. & Pardi, A. Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science 253, 191–194 (1991).

    Article  CAS  Google Scholar 

  19. Rüdisser, S. & Tinoco, I., Jr. Solution structure of cobalt(III)hexammine complexed to the GAAA tetraloop, and metal-ion binding to G·A mismatches. J. Mol. Biol. 295, 1211–1223 (2000).

    Article  Google Scholar 

  20. 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 

  21. Ferat, J.L. & Michel, F. Group II self-splicing introns in bacteria. Nature 364, 358–361 (1993).

    Article  CAS  Google Scholar 

  22. Chu, V.T., Adamidi, C., Liu, Q., Perlman, P.S. & Pyle, A.M. Control of branch-site choice by a group II intron. EMBO J. 20, 6866–6876 (2001).

    Article  CAS  Google Scholar 

  23. Boudvillain, M. & Pyle, A.M. Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis. EMBO J. 17, 7091–7104 (1998).

    Article  CAS  Google Scholar 

  24. Basu, S. et al. A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor. Nat. Struct. Biol. 5, 986–992 (1998).

    Article  CAS  Google Scholar 

  25. Schmidt, U., Podar, M., Stahl, U. & Perlman, P.S. Mutations of the two-nucleotide bulge of D5 of a group II intron block splicing in vitro and in vivo: phenotypes and suppressor mutations. RNA 2, 1161–1172 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Chanfreau, G. & Jacquier, A. An RNA conformational change between the two chemical steps of group II self-splicing. EMBO J. 15, 3466–3476 (1996).

    Article  CAS  Google Scholar 

  27. Chin, K., Sharp, K.A., Honig, B. & Pyle, A.M. Calculating the electrostatic properties of RNA provides new insights into molecular interactions and function. Nat. Struct. Biol. 6, 1055–1061 (1999).

    Article  CAS  Google Scholar 

  28. Schmitz, M. & Tinoco, I Jr. Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA. RNA 6, 1212–1225 (2000).

    Article  CAS  Google Scholar 

  29. Allain, F.H.-T. & Varani, G. Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns. Nucleic Acids Res. 23, 341–350 (1995).

    Article  CAS  Google Scholar 

  30. Duarte, C.M., Wadley, L. & 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  Google Scholar 

  31. Zhou, H.J., Vermeulen, A., Jucker, F.M. & Pardi, A. Incorporating residual dipolar couplings into the NMR solution structure determination of nucleic acids. Biopolymers 52, 168–180 (1999).

    Article  CAS  Google Scholar 

  32. Hansen, M.R., Hanson, P. & Pardi, A. Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions. Methods Enzymol. 317, 220–240 (2000).

    Article  CAS  Google Scholar 

  33. Guntert, P., Mumenthaler, C. & Wüthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997).

    Article  CAS  Google Scholar 

  34. Zweckstetter, M. & Bax, A. Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J. Am. Chem. Soc. 122, 3791–3792 (2000).

    Article  CAS  Google Scholar 

  35. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  36. Warren, J.J. & Moore, P.B. A maximum likelihood method for determining DaPQ and R for sets of dipolar coupling data. J. Magn. Res. 149, 271–275 (2001).

    Article  CAS  Google Scholar 

  37. Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55, 29–32 (1996).

    Article  CAS  Google Scholar 

  38. Al-Hashimi, H.M., Gorin, A., Majumdar, A., Gosser, Y. & Patel, D.J. Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings. J. Mol. Biol. 318, 637–649 (2002).

    Article  CAS  Google Scholar 

  39. Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296. (1991).

    Article  CAS  Google Scholar 

  40. 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  Google Scholar 

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Acknowledgements

We thank L. Nikstad for helping with structure determination and P. Pang, C. Duarte and L. Wadley for many helpful discussions. NMR studies carried out at the National Magnetic Resonance Facility at Madison were supported by the US National Institutes of Health (NIH) Biomedical Technology Program with additional equipment funding from the University of Wisconsin, US National Science Foundation (NSF) Academic Infrastructure Program, NIH Shared Instrumentation Program, NSF Biological Instrumentation Program, and the US Department of Agriculture. We are grateful for financial support from the Swiss Academy of Natural Sciences and the Swiss National Science Foundation (Grant for Young Scientists for R.K.O.S.), the NSF (A.G.P.) and the NIH (S.E.B. and A.M.P.). A.M.P. is a Howard Hughes Medical Institute investigator.

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Correspondence to Samuel E Butcher or Anna Marie Pyle.

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Sigel, R., Sashital, D., Abramovitz, D. et al. Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif. Nat Struct Mol Biol 11, 187–192 (2004). https://doi.org/10.1038/nsmb717

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