Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Dec 11;21(24):5656–5660. doi: 10.1093/nar/21.24.5656

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions.

S Sawata 1, T Shimayama 1, M Komiyama 1, P K Kumar 1, S Nishikawa 1, K Taira 1
PMCID: PMC310531  PMID: 7506830

Abstract

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.

Full text

PDF
5656

Images in this article

5658Image
on p.5658

Selected References

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

  1. 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]
  2. 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]
  3. Fedor M. J., Uhlenbeck O. C. Substrate sequence effects on "hammerhead" RNA catalytic efficiency. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1668–1672. doi: 10.1073/pnas.87.5.1668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hendry P., McCall M. J., Santiago F. S., Jennings P. A. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability. Nucleic Acids Res. 1992 Nov 11;20(21):5737–5741. doi: 10.1093/nar/20.21.5737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Krol A., Carbon P. A guide for probing native small nuclear RNA and ribonucleoprotein structures. Methods Enzymol. 1989;180:212–227. doi: 10.1016/0076-6879(89)80103-x. [DOI] [PubMed] [Google Scholar]
  8. McCall M. J., Hendry P., Jennings P. A. Minimal sequence requirements for ribozyme activity. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5710–5714. doi: 10.1073/pnas.89.13.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Perreault J. P., Labuda D., Usman N., Yang J. H., Cedergren R. Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis. Biochemistry. 1991 Apr 23;30(16):4020–4025. doi: 10.1021/bi00230a029. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Piccirilli J. A., Vyle J. S., Caruthers M. H., Cech T. R. Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature. 1993 Jan 7;361(6407):85–88. doi: 10.1038/361085a0. [DOI] [PubMed] [Google Scholar]
  12. Shimayama T., Nishikawa F., Nishikawa S., Taira K. Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages. Nucleic Acids Res. 1993 Jun 11;21(11):2605–2611. doi: 10.1093/nar/21.11.2605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shimayama T., Sawata S., Komiyama M., Takagi Y., Tanaka Y., Wada A., Sugimoto N., Rossi J. J., Nishikawa F., Nishikawa S. Substitution of non-catalytic stem and loop regions of hammerhead ribozyme with DNA counterparts only increases KM without sacrificing the catalytic step (kcat): a way to improve substrate-specificity. Nucleic Acids Symp Ser. 1992;(27):17–18. [PubMed] [Google Scholar]
  14. 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]
  15. Taylor N. R., Kaplan B. E., Swiderski P., Li H., Rossi J. J. Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo. Nucleic Acids Res. 1992 Sep 11;20(17):4559–4565. doi: 10.1093/nar/20.17.4559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Uchimaru T., Uebayasi M., Tanabe K., Taira K. Theoretical analyses on the role of Mg2+ ions in ribozyme reactions. FASEB J. 1993 Jan;7(1):137–142. doi: 10.1096/fasebj.7.1.8422960. [DOI] [PubMed] [Google Scholar]
  17. Uebayasi M., Uchimaru T., Tanabe K., Nishikawa S., Taira K. Preferential chelation of cationic ligands to axial-equatorial oxygens over equatorial-equatorial dianionic oxygens: implication to the mechanism of action of ribozymes. Nucleic Acids Symp Ser. 1991;(25):107–108. [PubMed] [Google Scholar]
  18. Yang J. H., Perreault J. P., Labuda D., Usman N., Cedergren R. Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme. Biochemistry. 1990 Dec 25;29(51):11156–11160. doi: 10.1021/bi00503a002. [DOI] [PubMed] [Google Scholar]
  19. 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]

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

RESOURCES