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. 2000 Feb;6(2):189–198. doi: 10.1017/s1355838200991441

Mg2+-independent hairpin ribozyme catalysis in hydrated RNA films.

A A Seyhan 1, J M Burke 1
PMCID: PMC1369905  PMID: 10688358

Abstract

The hairpin ribozyme catalyzes RNA cleavage in partially hydrated RNA films in the absence of added divalent cations. This reaction exhibits the characteristics associated with the RNA cleavage reaction observed under standard conditions in solution. Catalysis is a site-specific intramolecular transesterification reaction, requires the 2'-hydroxyl group of substrate nucleotide A(-1), and generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. Mutations in both ribozyme and substrate abolish catalysis in hydrated films. The reaction is accelerated by cations that may enhance binding, conformational stability, and catalytic activity, and is inhibited by Tb3+. The reaction has an apparent temperature optimum of 4 degrees C. At this temperature, cleavage is slow (k(obs): 2 d(-1)) and progressive, with accumulation of cleavage products to an extent of 40%. The use of synthetic RNAs, chelators, and analysis of all reaction components by inductively coupled plasma-optical spectrophotometry (ICPOES) effectively rules out the possibility of contaminating divalent metals in the reactions. Catalysis is minimal under conditions of extreme dehydration, indicating that the reaction requires hydration of RNA by atmospheric water. Our results provide a further caution for those studying the biochemical activity of ribozymes in vitro and in cells, as unanticipated catalysis could occur during RNA manipulation and lead to misinterpretation of data.

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Selected References

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  1. Affleck R., Xu Z. F., Suzawa V., Focht K., Clark D. S., Dordick J. S. Enzymatic catalysis and dynamics in low-water environments. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1100–1104. doi: 10.1073/pnas.89.3.1100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bone S., Pethig R. Dielectric studies of the binding of water to lysozyme. J Mol Biol. 1982 May 25;157(3):571–575. doi: 10.1016/0022-2836(82)90477-6. [DOI] [PubMed] [Google Scholar]
  3. Butcher S. E., Heckman J. E., Burke J. M. Reconstitution of hairpin ribozyme activity following separation of functional domains. J Biol Chem. 1995 Dec 15;270(50):29648–29651. doi: 10.1074/jbc.270.50.29648. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R. Self-splicing of group I introns. Annu Rev Biochem. 1990;59:543–568. doi: 10.1146/annurev.bi.59.070190.002551. [DOI] [PubMed] [Google Scholar]
  5. Chowrira B. M., Berzal-Herranz A., Burke J. M. Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme. Biochemistry. 1993 Feb 2;32(4):1088–1095. doi: 10.1021/bi00055a014. [DOI] [PubMed] [Google Scholar]
  6. Chowrira B. M., Berzal-Herranz A., Burke J. M. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature. 1991 Nov 28;354(6351):320–322. doi: 10.1038/354320a0. [DOI] [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. 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]
  9. 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]
  10. Earnshaw D. J., Gait M. J. Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions. Nucleic Acids Res. 1998 Dec 15;26(24):5551–5561. doi: 10.1093/nar/26.24.5551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Earnshaw D. J., Gait M. J. Progress toward the structure and therapeutic use of the hairpin ribozyme. Antisense Nucleic Acid Drug Dev. 1997 Aug;7(4):403–411. doi: 10.1089/oli.1.1997.7.403. [DOI] [PubMed] [Google Scholar]
  12. Esteban J. A., Banerjee A. R., Burke J. M. Kinetic mechanism of the hairpin ribozyme. Identification and characterization of two nonexchangeable conformations. J Biol Chem. 1997 May 23;272(21):13629–13639. doi: 10.1074/jbc.272.21.13629. [DOI] [PubMed] [Google Scholar]
  13. Esteban J. A., Walter N. G., Kotzorek G., Heckman J. E., Burke J. M. Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6091–6096. doi: 10.1073/pnas.95.11.6091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feig A. L., Scott W. G., Uhlenbeck O. C. Inhibition of the hammerhead ribozyme cleavage reaction by site-specific binding of Tb. Science. 1998 Jan 2;279(5347):81–84. doi: 10.1126/science.279.5347.81. [DOI] [PubMed] [Google Scholar]
  15. Feldstein P. A., Bruening G. Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA. Nucleic Acids Res. 1993 Apr 25;21(8):1991–1998. doi: 10.1093/nar/21.8.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Forster A. C., Jeffries A. C., Sheldon C. C., Symons R. H. Structural and ionic requirements for self-cleavage of virusoid RNAs and trans self-cleavage of viroid RNA. Cold Spring Harb Symp Quant Biol. 1987;52:249–259. doi: 10.1101/sqb.1987.052.01.030. [DOI] [PubMed] [Google Scholar]
  17. Geyer C. R., Sen D. Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme. Chem Biol. 1997 Aug;4(8):579–593. doi: 10.1016/s1074-5521(97)90244-1. [DOI] [PubMed] [Google Scholar]
  18. Hampel A., Cowan J. A. A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage. Chem Biol. 1997 Jul;4(7):513–517. doi: 10.1016/s1074-5521(97)90323-9. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Jaeger L., Westhof E., Michel F. Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA. J Mol Biol. 1991 Oct 20;221(4):1153–1164. doi: 10.1016/0022-2836(91)90925-v. [DOI] [PubMed] [Google Scholar]
  21. Long D. M., Uhlenbeck O. C. Self-cleaving catalytic RNA. FASEB J. 1993 Jan;7(1):25–30. doi: 10.1096/fasebj.7.1.8422971. [DOI] [PubMed] [Google Scholar]
  22. Maksareva E. Iu, Khurgin Iu I. Tverdofaznye fermentativnye reaktsii. V. Sravnenie kataliticheskikh svoistv subtilizina i alpha-khimotripsina v otsutstvie rastvoritelia. Bioorg Khim. 1995 Jan;21(1):24–27. [PubMed] [Google Scholar]
  23. Murray J. B., Seyhan A. A., Walter N. G., Burke J. M., Scott W. G. The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem Biol. 1998 Oct;5(10):587–595. doi: 10.1016/s1074-5521(98)90116-8. [DOI] [PubMed] [Google Scholar]
  24. Nesbitt S. M., Erlacher H. A., Fedor M. J. The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects. J Mol Biol. 1999 Mar 5;286(4):1009–1024. doi: 10.1006/jmbi.1999.2543. [DOI] [PubMed] [Google Scholar]
  25. Nesbitt S., Hegg L. A., Fedor M. J. An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis. Chem Biol. 1997 Aug;4(8):619–630. doi: 10.1016/s1074-5521(97)90247-7. [DOI] [PubMed] [Google Scholar]
  26. Porschke D., Burke J. M., Walter N. G. Global structure and flexibility of hairpin ribozymes with extended terminal helices. J Mol Biol. 1999 Jun 18;289(4):799–813. doi: 10.1006/jmbi.1999.2777. [DOI] [PubMed] [Google Scholar]
  27. Prody G. A., Bakos J. T., Buzayan J. M., Schneider I. R., Bruening G. Autolytic processing of dimeric plant virus satellite RNA. Science. 1986 Mar 28;231(4745):1577–1580. doi: 10.1126/science.231.4745.1577. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Seyhan A. A., Amaral J., Burke J. M. Intracellular RNA cleavage by the hairpin ribozyme. Nucleic Acids Res. 1998 Aug 1;26(15):3494–3504. doi: 10.1093/nar/26.15.3494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Suga H., Cowan J. A., Szostak J. W. Unusual metal ion catalysis in an acyl-transferase ribozyme. Biochemistry. 1998 Jul 14;37(28):10118–10125. doi: 10.1021/bi980432a. [DOI] [PubMed] [Google Scholar]
  31. Walter N. G., Burke J. M. Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates. RNA. 1997 Apr;3(4):392–404. [PMC free article] [PubMed] [Google Scholar]
  32. Walter N. G., Hampel K. J., Brown K. M., Burke J. M. Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer. EMBO J. 1998 Apr 15;17(8):2378–2391. doi: 10.1093/emboj/17.8.2378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Young K. J., Gill F., Grasby J. A. Metal ions play a passive role in the hairpin ribozyme catalysed reaction. Nucleic Acids Res. 1997 Oct 1;25(19):3760–3766. doi: 10.1093/nar/25.19.3760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zaks A., Klibanov A. M. Enzymatic catalysis in nonaqueous solvents. J Biol Chem. 1988 Mar 5;263(7):3194–3201. [PubMed] [Google Scholar]

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