Skip to main content
PMC home page
RNA logoLink to RNA
. 1998 Oct;4(10):1251–1258. doi: 10.1017/s1355838298980943

Cryoenzymology of the hammerhead ribozyme.

A L Feig 1, G E Ammons 1, O C Uhlenbeck 1
PMCID: PMC1369697  PMID: 9769099

Abstract

The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and -33 degrees C. Eyring plots are linear down to -27 degrees C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.

Full Text

The Full Text of this article is available as a PDF (146.4 KB).

Selected References

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

  1. 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]
  2. Bassi G. S., Murchie A. I., Walter F., Clegg R. M., Lilley D. M. Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study. EMBO J. 1997 Dec 15;16(24):7481–7489. doi: 10.1093/emboj/16.24.7481. [DOI] [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. Breslauer K. J., Bodnar C. M., McCarthy J. E. The role of solvents in the stabilization of helical structure: the low pH ribo A8 and A10 double helices in mixed solvents. Biophys Chem. 1978 Nov;9(1):71–78. doi: 10.1016/0301-4622(78)87017-3. [DOI] [PubMed] [Google Scholar]
  5. Burgin A. B., Jr, Gonzalez C., Matulic-Adamic J., Karpeisky A. M., Usman N., McSwiggen J. A., Beigelman L. Chemically modified hammerhead ribozymes with improved catalytic rates. Biochemistry. 1996 Nov 12;35(45):14090–14097. doi: 10.1021/bi961264u. [DOI] [PubMed] [Google Scholar]
  6. Clouet-D'Orval B., Uhlenbeck O. C. Kinetic characterization of two I/II format hammerhead ribozymes. RNA. 1996 May;2(5):483–491. [PMC free article] [PubMed] [Google Scholar]
  7. Clouet-d'Orval B., Uhlenbeck O. C. Hammerhead ribozymes with a faster cleavage rate. Biochemistry. 1997 Jul 29;36(30):9087–9092. doi: 10.1021/bi9710941. [DOI] [PubMed] [Google Scholar]
  8. Douzou P. Cryoenzymology. Cryobiology. 1983 Oct;20(5):625–635. doi: 10.1016/0011-2240(83)90050-0. [DOI] [PubMed] [Google Scholar]
  9. Fedor M. J., Uhlenbeck O. C. Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry. 1992 Dec 8;31(48):12042–12054. doi: 10.1021/bi00163a012. [DOI] [PubMed] [Google Scholar]
  10. Fink A. L. Cryoenzymology: the use of sub-zero temperatures and fluid solutions in the study of enzyme mechanisms. J Theor Biol. 1976 Sep 21;61(2):419–445. doi: 10.1016/0022-5193(76)90028-x. [DOI] [PubMed] [Google Scholar]
  11. Fink A. L., Geeves M. A. Cryoenzymology: the study of enzyme catalysis at subzero temperatures. Methods Enzymol. 1979;63:336–370. doi: 10.1016/0076-6879(79)63015-x. [DOI] [PubMed] [Google Scholar]
  12. Gardiner K. J., Marsh T. L., Pace N. R. Ion dependence of the Bacillus subtilis RNase P reaction. J Biol Chem. 1985 May 10;260(9):5415–5419. [PubMed] [Google Scholar]
  13. Hanna M., Szostak J. W. Suppression of mutations in the core of the Tetrahymena ribozyme by spermidine, ethanol and by substrate stabilization. Nucleic Acids Res. 1994 Dec 11;22(24):5326–5331. doi: 10.1093/nar/22.24.5326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hertel K. J., Herschlag D., Uhlenbeck O. C. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry. 1994 Mar 22;33(11):3374–3385. doi: 10.1021/bi00177a031. [DOI] [PubMed] [Google Scholar]
  15. Hertel K. J., Herschlag D., Uhlenbeck O. C. Specificity of hammerhead ribozyme cleavage. EMBO J. 1996 Jul 15;15(14):3751–3757. [PMC free article] [PubMed] [Google Scholar]
  16. Hertel K. J., Uhlenbeck O. C. The internal equilibrium of the hammerhead ribozyme reaction. Biochemistry. 1995 Feb 7;34(5):1744–1749. doi: 10.1021/bi00005a031. [DOI] [PubMed] [Google Scholar]
  17. Heus H. A., Pardi A. Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence. J Mol Biol. 1991 Jan 5;217(1):113–124. doi: 10.1016/0022-2836(91)90615-d. [DOI] [PubMed] [Google Scholar]
  18. Limmer S. Mismatch base pairs in RNA. Prog Nucleic Acid Res Mol Biol. 1997;57:1–39. doi: 10.1016/s0079-6603(08)60276-7. [DOI] [PubMed] [Google Scholar]
  19. Makinen M. W., Fink A. L. Reactivity and cryoenzymology of enzymes in the crystalline state. Annu Rev Biophys Bioeng. 1977;6:301–343. doi: 10.1146/annurev.bb.06.060177.001505. [DOI] [PubMed] [Google Scholar]
  20. Makinen M. W., Kuo L. C., Dymowski J. J., Jaffer S. Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis. J Biol Chem. 1979 Jan 25;254(2):356–366. [PubMed] [Google Scholar]
  21. McKay D. B. Structure and function of the hammerhead ribozyme: an unfinished story. RNA. 1996 May;2(5):395–403. [PMC free article] [PubMed] [Google Scholar]
  22. 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. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. More N., Daniel R. M., Petach H. H. The effect of low temperatures on enzyme activity. Biochem J. 1995 Jan 1;305(Pt 1):17–20. doi: 10.1042/bj3050017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Peracchi A., Beigelman L., Scott E. C., Uhlenbeck O. C., Herschlag D. Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation. J Biol Chem. 1997 Oct 24;272(43):26822–26826. doi: 10.1074/jbc.272.43.26822. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Prinz H., Maelicke A., Cramer F. Unfolding of yeast transfer ribonucleic acid species caused by addition of organic solvents and studied by circular dichroism. Biochemistry. 1974 Mar 26;13(7):1322–1326. doi: 10.1021/bi00704a003. [DOI] [PubMed] [Google Scholar]
  28. Rasmussen B. F., Stock A. M., Ringe D., Petsko G. A. Crystalline ribonuclease A loses function below the dynamical transition at 220 K. Nature. 1992 Jun 4;357(6377):423–424. doi: 10.1038/357423a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Schweizer M. P. PMR studies of organic solvent denatured transfer RNA. Biochem Biophys Res Commun. 1969 Sep 10;36(6):871–877. doi: 10.1016/0006-291x(69)90284-8. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Simorre J. P., Legault P., Baidya N., Uhlenbeck O. C., Maloney L., Wincott F., Usman N., Beigelman L., Pardi A. Structural variation induced by different nucleotides at the cleavage site of the hammerhead ribozyme. Biochemistry. 1998 Mar 24;37(12):4034–4044. doi: 10.1021/bi972493z. [DOI] [PubMed] [Google Scholar]
  35. Simorre J. P., Legault P., Hangar A. B., Michiels P., Pardi A. A conformational change in the catalytic core of the hammerhead ribozyme upon cleavage of an RNA substrate. Biochemistry. 1997 Jan 21;36(3):518–525. doi: 10.1021/bi9620520. [DOI] [PubMed] [Google Scholar]
  36. Stage-Zimmermann T. K., Uhlenbeck O. C. Hammerhead ribozyme kinetics. RNA. 1998 Aug;4(8):875–889. doi: 10.1017/s1355838298980876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Takagi Y., Taira K. Temperature-dependent change in the rate-determining step in a reaction catalyzed by a hammerhead ribozyme. FEBS Lett. 1995 Mar 20;361(2-3):273–276. doi: 10.1016/0014-5793(95)00192-c. [DOI] [PubMed] [Google Scholar]
  38. Travers F., Barman T. Cryoenzymology: how to practice kinetic and structural studies. Biochimie. 1995;77(12):937–948. doi: 10.1016/0300-9084(95)80005-0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Zhou De-Min, Taira Kazunari. The Hydrolysis of RNA: From Theoretical Calculations to the Hammerhead Ribozyme-Mediated Cleavage of RNA. Chem Rev. 1998 May 7;98(3):991–1026. doi: 10.1021/cr9604292. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES