Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Aug 15;25(16):3297–3301. doi: 10.1093/nar/25.16.3297

A new method to monitor the rate of conformational transitions in RNA.

E J Maglott 1, G D Glick 1
PMCID: PMC146897  PMID: 9241244

Abstract

Many RNAs need Mg2+to produce stable tertiary structures. Here we describe a simple method to measure the rate and activation parameters of tertiary structure unfolding that exploits this Mg2+dependence. Our approach is based on mixing an RNA solution with excess EDTA in a stopped-flow instrument equipped with an absorbance detector, under conditions of temperature and ionic strength where, after chelation of Mg2+, tertiary structure unfolds. We have demonstrated the utility of this method by studying phenylalanine-specific transfer RNA from yeast (tRNAPhe) because the unfolding rates and the corresponding activation parameters have been determined previously and provide a benchmark for our technique. We find that within error, our stopped-flow method reproduces both the rate and activation enthalpy for tertiary unfolding of yeast tRNAPhe measured previously by temperature-jump relaxation kinetics. Since many different RNAs require divalent magnesium for tertiary structure stabilization, this technique should be applicable to study the folding of other RNAs.

Full Text

The Full Text of this article is available as a PDF (80.2 KB).

Selected References

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

  1. Banerjee A. R., Jaeger J. A., Turner D. H. Thermal unfolding of a group I ribozyme: the low-temperature transition is primarily disruption of tertiary structure. Biochemistry. 1993 Jan 12;32(1):153–163. doi: 10.1021/bi00052a021. [DOI] [PubMed] [Google Scholar]
  2. Banerjee A. R., Turner D. H. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry. 1995 May 16;34(19):6504–6512. doi: 10.1021/bi00019a031. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bevilacqua P. C., Li Y., Turner D. H. Fluorescence-detected stopped flow with a pyrene labeled substrate reveals that guanosine facilitates docking of the 5' cleavage site into a high free energy binding mode in the Tetrahymena ribozyme. Biochemistry. 1994 Sep 20;33(37):11340–11348. doi: 10.1021/bi00203a032. [DOI] [PubMed] [Google Scholar]
  5. Butcher S. E., Burke J. M. Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex. J Mol Biol. 1994 Nov 18;244(1):52–63. doi: 10.1006/jmbi.1994.1703. [DOI] [PubMed] [Google Scholar]
  6. Cate J. H., Gooding A. R., Podell E., Zhou K., Golden B. L., Kundrot C. E., Cech T. R., Doudna J. A. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science. 1996 Sep 20;273(5282):1678–1685. doi: 10.1126/science.273.5282.1678. [DOI] [PubMed] [Google Scholar]
  7. Celander D. W., Cech T. R. Visualizing the higher order folding of a catalytic RNA molecule. Science. 1991 Jan 25;251(4992):401–407. doi: 10.1126/science.1989074. [DOI] [PubMed] [Google Scholar]
  8. Cheong C., Varani G., Tinoco I., Jr Solution structure of an unusually stable RNA hairpin, 5'GGAC(UUCG)GUCC. Nature. 1990 Aug 16;346(6285):680–682. doi: 10.1038/346680a0. [DOI] [PubMed] [Google Scholar]
  9. Christian E. L., Yarus M. Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena. Biochemistry. 1993 May 4;32(17):4475–4480. doi: 10.1021/bi00068a001. [DOI] [PubMed] [Google Scholar]
  10. Cole P. E., Yang S. K., Crothers D. M. Conformational changes of transfer ribonucleic acid. Equilibrium phase diagrams. Biochemistry. 1972 Nov 7;11(23):4358–4368. doi: 10.1021/bi00773a024. [DOI] [PubMed] [Google Scholar]
  11. Coutts S. M., Riesner D., Römer R., Rabl C. R., Maass G. Kinetics of conformational changes in tRNA Phe (yeast) as studied by the fluorescence of the Y-base and of formycin substituted for the 3'-terminal adenine. Biophys Chem. 1975 Oct;3(4):275–289. doi: 10.1016/0301-4622(75)80020-2. [DOI] [PubMed] [Google Scholar]
  12. Crothers D. M., Cole P. E., Hilbers C. W., Shulman R. G. The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA. J Mol Biol. 1974 Jul 25;87(1):63–88. doi: 10.1016/0022-2836(74)90560-9. [DOI] [PubMed] [Google Scholar]
  13. Downs W. D., Cech T. R. Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks. RNA. 1996 Jul;2(7):718–732. [PMC free article] [PubMed] [Google Scholar]
  14. Draper D. E., Xing Y., Laing L. G. Thermodynamics of RNA unfolding: stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion. J Mol Biol. 1995 Jun 2;249(2):231–238. doi: 10.1006/jmbi.1995.0291. [DOI] [PubMed] [Google Scholar]
  15. Emerick V. L., Pan J., Woodson S. A. Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron. Biochemistry. 1996 Oct 15;35(41):13469–13477. doi: 10.1021/bi960865i. [DOI] [PubMed] [Google Scholar]
  16. Emerick V. L., Woodson S. A. Fingerprinting the folding of a group I precursor RNA. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9675–9679. doi: 10.1073/pnas.91.21.9675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fresco J. R., Adams A., Ascione R., Henley D., Lindahl T. Tertiary structure in transfer ribonucleic acids. Cold Spring Harb Symp Quant Biol. 1966;31:527–537. doi: 10.1101/sqb.1966.031.01.068. [DOI] [PubMed] [Google Scholar]
  18. Guéron M., Leroy J. L. Significance and mechanism of divalent-ion binding to transfer RNA. Biophys J. 1982 Jun;38(3):231–236. doi: 10.1016/S0006-3495(82)84553-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hinz H. J., Filimonov V. V., Privalov P. L. Calorimetric studies on melting of tRNA Phe (yeast). Eur J Biochem. 1977 Jan 3;72(1):79–86. doi: 10.1111/j.1432-1033.1977.tb11226.x. [DOI] [PubMed] [Google Scholar]
  20. Hyman E. S. A study of the rate of chelation of magnesium by CDTA and EDTA in the ATP (NA+ + K+)-ATPase system. Biochim Biophys Acta. 1980 Aug 4;600(2):553–570. doi: 10.1016/0005-2736(80)90456-3. [DOI] [PubMed] [Google Scholar]
  21. Jaeger L., Westhof E., Michel F. Monitoring of the cooperative unfolding of the sunY group I intron of bacteriophage T4. The active form of the sunY ribozyme is stabilized by multiple interactions with 3' terminal intron components. J Mol Biol. 1993 Nov 20;234(2):331–346. doi: 10.1006/jmbi.1993.1590. [DOI] [PubMed] [Google Scholar]
  22. Johnston P. D., Redfield A. G. Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance. Biochemistry. 1981 Jul 7;20(14):3996–4006. doi: 10.1021/bi00517a008. [DOI] [PubMed] [Google Scholar]
  23. Kazakov S., Altman S. Site-specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9193–9197. doi: 10.1073/pnas.88.20.9193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laing L. G., Gluick T. C., Draper D. E. Stabilization of RNA structure by Mg ions. Specific and non-specific effects. J Mol Biol. 1994 Apr 15;237(5):577–587. doi: 10.1006/jmbi.1994.1256. [DOI] [PubMed] [Google Scholar]
  25. Leroy J. L., Guéron M. Electrostatic effects in divalent ion binding to tRNA. Biopolymers. 1977 Nov;16(11):2429–2446. doi: 10.1002/bip.1977.360161108. [DOI] [PubMed] [Google Scholar]
  26. Leroy J. L., Guéron M., Thomas G., Favre A. Role of divalent ions in folding of tRNA. Eur J Biochem. 1977 Apr 15;74(3):567–574. doi: 10.1111/j.1432-1033.1977.tb11426.x. [DOI] [PubMed] [Google Scholar]
  27. Levy J., Rialdi G., Biltonen R. Thermodynamic studies of transfer ribonucleic acids. II. Characterization of the thermal unfolding of yeast phenylalanine-specific transfer ribonucleic acid. Biochemistry. 1972 Oct 24;11(22):4138–4144. doi: 10.1021/bi00772a017. [DOI] [PubMed] [Google Scholar]
  28. Lorsch J. R., Szostak J. W. Chance and necessity in the selection of nucleic acid catalysts. Acc Chem Res. 1996 Feb;29(2):103–110. doi: 10.1021/ar9501378. [DOI] [PubMed] [Google Scholar]
  29. Lu M., Draper D. E. Bases defining an ammonium and magnesium ion-dependent tertiary structure within the large subunit ribosomal RNA. J Mol Biol. 1994 Dec 16;244(5):572–585. doi: 10.1006/jmbi.1994.1753. [DOI] [PubMed] [Google Scholar]
  30. Matouschek A., Fersht A. R. Protein engineering in analysis of protein folding pathways and stability. Methods Enzymol. 1991;202:82–112. doi: 10.1016/0076-6879(91)02008-w. [DOI] [PubMed] [Google Scholar]
  31. Matouschek A., Kellis J. T., Jr, Serrano L., Bycroft M., Fersht A. R. Transient folding intermediates characterized by protein engineering. Nature. 1990 Aug 2;346(6283):440–445. doi: 10.1038/346440a0. [DOI] [PubMed] [Google Scholar]
  32. Narlikar G. J., Herschlag D. Isolation of a local tertiary folding transition in the context of a globally folded RNA. Nat Struct Biol. 1996 Aug;3(8):701–710. doi: 10.1038/nsb0896-701. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Privalov P. L., Filimonov V. V. Thermodynamic analysis of transfer RNA unfolding. J Mol Biol. 1978 Jul 15;122(4):447–464. doi: 10.1016/0022-2836(78)90421-7. [DOI] [PubMed] [Google Scholar]
  35. Pyle A. M., Green J. B. RNA folding. Curr Opin Struct Biol. 1995 Jun;5(3):303–310. doi: 10.1016/0959-440x(95)80091-3. [DOI] [PubMed] [Google Scholar]
  36. Pyle A. M. Ribozymes: a distinct class of metalloenzymes. Science. 1993 Aug 6;261(5122):709–714. doi: 10.1126/science.7688142. [DOI] [PubMed] [Google Scholar]
  37. 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. 1997 Apr 22;36(16):4718–4730. doi: 10.1021/bi962665c. [DOI] [PubMed] [Google Scholar]
  38. Reid S. S., Cowan J. A. Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity. Biochemistry. 1990 Jun 26;29(25):6025–6032. doi: 10.1021/bi00477a021. [DOI] [PubMed] [Google Scholar]
  39. Riesner D., Maass G., Thiebe R., Philippsen P., Zachau H. G. The conformational transitions in yeast tRNAPhe as studied with tRNAPhe fragments. Eur J Biochem. 1973 Jul 2;36(1):76–88. doi: 10.1111/j.1432-1033.1973.tb02887.x. [DOI] [PubMed] [Google Scholar]
  40. Riesner D., Römer R., Maass G. Kinetic study of the three conformational transitions of alanine specific transfer RNA from yeast. Eur J Biochem. 1970 Jul;15(1):85–91. doi: 10.1111/j.1432-1033.1970.tb00979.x. [DOI] [PubMed] [Google Scholar]
  41. Römer R., Riesner D., Maass G. Resolution of five conformational transitions in phenylalaninespecific tRNA from yeast. FEBS Lett. 1970 Oct;10(5):352–357. doi: 10.1016/0014-5793(70)80471-9. [DOI] [PubMed] [Google Scholar]
  42. Sclavi B., Woodson S., Sullivan M., Chance M. R., Brenowitz M. Time-resolved synchrotron X-ray "footprinting", a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding. J Mol Biol. 1997 Feb 14;266(1):144–159. doi: 10.1006/jmbi.1996.0775. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Shen L. X., Cai Z., Tinoco I., Jr RNA structure at high resolution. FASEB J. 1995 Aug;9(11):1023–1033. doi: 10.1096/fasebj.9.11.7544309. [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. Urbanke C., Römer R., Maass G. Tertiary structure of tRNAPhe (yeast): kinetics and electrostatic repulsion. Eur J Biochem. 1975 Jul 1;55(2):439–444. doi: 10.1111/j.1432-1033.1975.tb02180.x. [DOI] [PubMed] [Google Scholar]
  47. Urbanke C., Römer R., Maass G. The binding of ethidium bromide to different conformations of tRNA. Unfolding of tertiary structure. Eur J Biochem. 1973 Mar 15;33(3):511–516. doi: 10.1111/j.1432-1033.1973.tb02710.x. [DOI] [PubMed] [Google Scholar]
  48. Walters J. A., Geerdes H. A., Hilbers C. W. On the binding of Mg2+ and Mn2+ to tRNA. Biophys Chem. 1977 Sep;7(2):147–151. doi: 10.1016/0301-4622(77)80007-0. [DOI] [PubMed] [Google Scholar]
  49. Williamson J. R. RNA origami. Nat Struct Biol. 1994 May;1(5):270–272. doi: 10.1038/nsb0594-270. [DOI] [PubMed] [Google Scholar]
  50. Yang S. K., Crothers D. M. Conformational changes of transfer ribonucleic acid. Comparison of the early melting transition of two tyrosine-specific transfer ribonucleic acids. Biochemistry. 1972 Nov 7;11(23):4375–4381. doi: 10.1021/bi00773a026. [DOI] [PubMed] [Google Scholar]
  51. Zarrinkar P. P., Wang J., Williamson J. R. Slow folding kinetics of RNase P RNA. RNA. 1996 Jun;2(6):564–573. [PMC free article] [PubMed] [Google Scholar]
  52. Zarrinkar P. P., Williamson J. R. Kinetic intermediates in RNA folding. Science. 1994 Aug 12;265(5174):918–924. doi: 10.1126/science.8052848. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES