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. 1997 Dec 15;16(24):7481–7489. doi: 10.1093/emboj/16.24.7481

Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study.

G S Bassi 1, A I Murchie 1, F Walter 1, R M Clegg 1, D M Lilley 1
PMCID: PMC1170347  PMID: 9405376

Abstract

The ion-induced folding transitions of the hammerhead ribozyme have been analysed by fluorescence resonance energy transfer. The hammerhead ribozyme may be regarded as a special example of a three-way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end-to-end vectors as a function of magnesium ion concentration. The data support two sequential ion-dependent transitions, which can be interpreted in the light of the crystal structures of the hammerhead ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000-10 000 M-1. The second structural transition corresponds to the formation of the catalytic domain of the ribozyme, induced by a single magnesium ion with an apparent association constant of approximately 1100 M-1. The hammerhead ribozyme provides a well-defined example of ion-dependent folding in RNA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amiri K. M., Hagerman P. J. Global conformation of a self-cleaving hammerhead RNA. Biochemistry. 1994 Nov 15;33(45):13172–13177. doi: 10.1021/bi00249a003. [DOI] [PubMed] [Google Scholar]
  2. Bassi G. S., Murchie A. I., Lilley D. M. The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation. RNA. 1996 Aug;2(8):756–768. [PMC free article] [PubMed] [Google Scholar]
  3. Bassi G. S., Møllegaard N. E., Murchie A. I., von Kitzing E., Lilley D. M. Ionic interactions and the global conformations of the hammerhead ribozyme. Nat Struct Biol. 1995 Jan;2(1):45–55. doi: 10.1038/nsb0195-45. [DOI] [PubMed] [Google Scholar]
  4. Clegg R. M. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 1992;211:353–388. doi: 10.1016/0076-6879(92)11020-j. [DOI] [PubMed] [Google Scholar]
  5. Clegg R. M., Murchie A. I., Zechel A., Carlberg C., Diekmann S., Lilley D. M. Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction. Biochemistry. 1992 May 26;31(20):4846–4856. doi: 10.1021/bi00135a016. [DOI] [PubMed] [Google Scholar]
  6. Clegg R. M., Murchie A. I., Zechel A., Lilley D. M. Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2994–2998. doi: 10.1073/pnas.90.7.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahm S. C., Derrick W. B., Uhlenbeck O. C. Evidence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. Biochemistry. 1993 Dec 7;32(48):13040–13045. doi: 10.1021/bi00211a013. [DOI] [PubMed] [Google Scholar]
  8. Dahm S. C., Uhlenbeck O. C. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry. 1991 Oct 1;30(39):9464–9469. doi: 10.1021/bi00103a011. [DOI] [PubMed] [Google Scholar]
  9. Duckett D. R., Murchie A. I., Diekmann S., von Kitzing E., Kemper B., Lilley D. M. The structure of the Holliday junction, and its resolution. Cell. 1988 Oct 7;55(1):79–89. doi: 10.1016/0092-8674(88)90011-6. [DOI] [PubMed] [Google Scholar]
  10. Duckett D. R., Murchie A. I., Lilley D. M. The global folding of four-way helical junctions in RNA, including that in U1 snRNA. Cell. 1995 Dec 15;83(6):1027–1036. doi: 10.1016/0092-8674(95)90218-x. [DOI] [PubMed] [Google Scholar]
  11. Epstein L. M., Gall J. G. Self-cleaving transcripts of satellite DNA from the newt. Cell. 1987 Feb 13;48(3):535–543. doi: 10.1016/0092-8674(87)90204-2. [DOI] [PubMed] [Google Scholar]
  12. Feldstein P. A., Buzayan J. M., Bruening G. Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA. Gene. 1989 Oct 15;82(1):53–61. doi: 10.1016/0378-1119(89)90029-2. [DOI] [PubMed] [Google Scholar]
  13. Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
  14. Fu D. J., McLaughlin L. W. Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding. Biochemistry. 1992 Nov 17;31(45):10941–10949. doi: 10.1021/bi00160a001. [DOI] [PubMed] [Google Scholar]
  15. Fu D. J., McLaughlin L. W. Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3985–3989. doi: 10.1073/pnas.89.9.3985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fu D. J., Rajur S. B., McLaughlin L. W. Activity of the hammerhead ribozyme upon inversion of the stereocenters for the guanosine 2'-hydroxyls. Biochemistry. 1994 Nov 22;33(46):13903–13909. doi: 10.1021/bi00250a045. [DOI] [PubMed] [Google Scholar]
  17. Fu D. J., Rajur S. B., McLaughlin L. W. Importance of specific guanosine N7-nitrogens and purine amino groups for efficient cleavage by a hammerhead ribozyme. Biochemistry. 1993 Oct 12;32(40):10629–10637. doi: 10.1021/bi00091a013. [DOI] [PubMed] [Google Scholar]
  18. Gast F. U., Amiri K. M., Hagerman P. J. Interhelix geometry of stems I and II of a self-cleaving hammerhead RNA. Biochemistry. 1994 Feb 22;33(7):1788–1796. doi: 10.1021/bi00173a023. [DOI] [PubMed] [Google Scholar]
  19. Grasby J. A., Jonathan P., Butler G., Gait M. J. The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage. Nucleic Acids Res. 1993 Sep 25;21(19):4444–4450. doi: 10.1093/nar/21.19.4444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hampel A., Tritz R. RNA catalytic properties of the minimum (-)sTRSV sequence. Biochemistry. 1989 Jun 13;28(12):4929–4933. doi: 10.1021/bi00438a002. [DOI] [PubMed] [Google Scholar]
  21. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  22. Hertel K. J., Pardi A., Uhlenbeck O. C., Koizumi M., Ohtsuka E., Uesugi S., Cedergren R., Eckstein F., Gerlach W. L., Hodgson R. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. doi: 10.1093/nar/20.12.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hutchins C. J., Rathjen P. D., Forster A. C., Symons R. H. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 1986 May 12;14(9):3627–3640. doi: 10.1093/nar/14.9.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klement R., Soumpasis D. M., Jovin T. M. Computation of ionic distributions around charged biomolecular structures: results for right-handed and left-handed DNA. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4631–4635. doi: 10.1073/pnas.88.11.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Knöll R., Bald R., Fürste J. P. Complete identification of nonbridging phosphate oxygens involved in hammerhead cleavage. RNA. 1997 Feb;3(2):132–140. [PMC free article] [PubMed] [Google Scholar]
  26. Koizumi M., Ohtsuka E. Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding. Biochemistry. 1991 May 28;30(21):5145–5150. doi: 10.1021/bi00235a005. [DOI] [PubMed] [Google Scholar]
  27. Lilley D. M., Clegg R. M., Diekmann S., Seeman N. C., von Kitzing E., Hagerman P. Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). A nomenclature of junctions and branchpoints in nucleic acids. Recommendations 1994. Eur J Biochem. 1995 May 15;230(1):1–2. doi: 10.1111/j.1432-1033.1995.tb20526.x. [DOI] [PubMed] [Google Scholar]
  28. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  29. Menger M., Tuschl T., Eckstein F., Porschke D. Mg(2+)-dependent conformational changes in the hammerhead ribozyme. Biochemistry. 1996 Nov 26;35(47):14710–14716. doi: 10.1021/bi960440w. [DOI] [PubMed] [Google Scholar]
  30. Murchie A. I., Clegg R. M., von Kitzing E., Duckett D. R., Diekmann S., Lilley D. M. Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature. 1989 Oct 26;341(6244):763–766. doi: 10.1038/341763a0. [DOI] [PubMed] [Google Scholar]
  31. Olsen D. B., Benseler F., Aurup H., Pieken W. A., Eckstein F. Study of a hammerhead ribozyme containing 2'-modified adenosine residues. Biochemistry. 1991 Oct 8;30(40):9735–9741. doi: 10.1021/bi00104a024. [DOI] [PubMed] [Google Scholar]
  32. Paolella G., Sproat B. S., Lamond A. I. Nuclease resistant ribozymes with high catalytic activity. EMBO J. 1992 May;11(5):1913–1919. doi: 10.1002/j.1460-2075.1992.tb05244.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Perreault J. P., Wu T. F., Cousineau B., Ogilvie K. K., Cedergren R. Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity. Nature. 1990 Apr 5;344(6266):565–567. doi: 10.1038/344565a0. [DOI] [PubMed] [Google Scholar]
  34. Pley H. W., Flaherty K. M., McKay D. B. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994 Nov 3;372(6501):68–74. doi: 10.1038/372068a0. [DOI] [PubMed] [Google Scholar]
  35. Ruffner D. E., Stormo G. D., Uhlenbeck O. C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry. 1990 Nov 27;29(47):10695–10702. doi: 10.1021/bi00499a018. [DOI] [PubMed] [Google Scholar]
  36. Ruffner D. E., Uhlenbeck O. C. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 1990 Oct 25;18(20):6025–6029. doi: 10.1093/nar/18.20.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scott W. G., Finch J. T., Klug A. The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell. 1995 Jun 30;81(7):991–1002. doi: 10.1016/s0092-8674(05)80004-2. [DOI] [PubMed] [Google Scholar]
  38. Scott W. G., Murray J. B., Arnold J. R., Stoddard B. L., Klug A. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science. 1996 Dec 20;274(5295):2065–2069. doi: 10.1126/science.274.5295.2065. [DOI] [PubMed] [Google Scholar]
  39. Sharmeen L., Kuo M. Y., Dinter-Gottlieb G., Taylor J. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J Virol. 1988 Aug;62(8):2674–2679. doi: 10.1128/jvi.62.8.2674-2679.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sigurdsson S. T., Tuschl T., Eckstein F. Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme. RNA. 1995 Aug;1(6):575–583. [PMC free article] [PubMed] [Google Scholar]
  41. Slim G., Gait M. J. Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 1991 Mar 25;19(6):1183–1188. doi: 10.1093/nar/19.6.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stein A., Crothers D. M. Equilibrium binding of magnesium(II) by Escherichia coli tRNAfMet. Biochemistry. 1976 Jan 13;15(1):157–160. doi: 10.1021/bi00646a024. [DOI] [PubMed] [Google Scholar]
  43. Tuschl T., Gohlke C., Jovin T. M., Westhof E., Eckstein F. A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements. Science. 1994 Nov 4;266(5186):785–789. doi: 10.1126/science.7973630. [DOI] [PubMed] [Google Scholar]
  44. Tuschl T., Ng M. M., Pieken W., Benseler F., Eckstein F. Importance of exocyclic base functional groups of central core guanosines for hammerhead ribozyme activity. Biochemistry. 1993 Nov 2;32(43):11658–11668. doi: 10.1021/bi00094a023. [DOI] [PubMed] [Google Scholar]
  45. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  46. Welch J. B., Walter F., Lilley D. M. Two inequivalent folding isomers of the three-way DNA junction with unpaired bases: sequence-dependence of the folded conformation. J Mol Biol. 1995 Aug 25;251(4):507–519. doi: 10.1006/jmbi.1995.0452. [DOI] [PubMed] [Google Scholar]
  47. Williams D. M., Pieken W. A., Eckstein F. Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):918–921. doi: 10.1073/pnas.89.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yang J. H., Usman N., Chartrand P., Cedergren R. Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. Biochemistry. 1992 Jun 2;31(21):5005–5009. doi: 10.1021/bi00136a013. [DOI] [PubMed] [Google Scholar]
  49. van Tol H., Buzayan J. M., Feldstein P. A., Eckstein F., Bruening G. Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration. Nucleic Acids Res. 1990 Apr 25;18(8):1971–1975. doi: 10.1093/nar/18.8.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]

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