Skip to main content

Defining Optimum Reaction Conditions for Hammerhead Ribozymes

  • Protocol
Ribozyme Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 74))

  • 586 Accesses

Abstract

Hammerhead ribozymes, in their trans-cleaving form catalyze the cleavage of complementary RNA strands, which contain the UX sequence (where X is A, U, or C) Immediately 5′ to the site of cleavage, although not all such sequences are cleaved with the same efficiency (1,2). The reactivity of the hammerhead ribozyme is assumed to follow the model outlined in Scheme 1, i.e., ribozyme and substrate anneal, cleavage takes place, and then dissociation of the two products occurs independently. The in vitro study of hammerhead activity allows manipulation of the reaction conditions in order to gain insight into the detail of the cleavage reaction.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Perriman, R., Delves, A., and Gerlach, W. L. (1992) Extended target-site specificity for a hammerhead ribozyme. Gene 113, 157–163.

    Article  PubMed  CAS  Google Scholar 

  2. Shimayama, T., Nishikawa, S., and Taira, K. (1995) Generality of the NUX rule kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes. Biochemistry 34, 3649–3654.

    Article  PubMed  CAS  Google Scholar 

  3. Perreault, J.-P., Labuda, D., Usman, N., Yang, J.-H., and Cedergren, R. (1991) Relationship between 2′-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis. Biochemistry 30, 4020–4025.

    Article  PubMed  CAS  Google Scholar 

  4. Uhlenbeck, O. C. (1987) A small catalytic oligoribonucleotide. Nature 328, 596–600.

    Article  PubMed  CAS  Google Scholar 

  5. Dahm, S. C. and Uhlenbeck, O. C. (1991) Role of divalent metal tons in the hammerhead RNA cleavage reaction. Biochemistry 30, 9464–9469.

    Article  PubMed  CAS  Google Scholar 

  6. Dahm, S. C., Derrick, W. B., and Uhlenbeck, O. C. (1993) Evidence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. Biochemistry 32, 13,040–13,045.

    Article  PubMed  CAS  Google Scholar 

  7. Takagi, Y. and Taira, K. (1995) Temperature-dependent change in the rate-determining step in a reaction catalyzed by a hammerhead ribozyme. FEBS Lett. 361, 273–276.

    Article  PubMed  CAS  Google Scholar 

  8. Hendry, P. and McCall, M. J. (1995) A comparison of the in vitro activity of DNA-armed and all-RNA hammerhead ribozymes. Nucleic Acids Rex. 23, 3928–3936.

    Article  CAS  Google Scholar 

  9. Buzayan, J. M., Gerlach, W. L., and Bruening, G. (1986) Non-enzymic cleavage and ligation of RNAs complementary to a plant virus satellite RNA. Nature 323, 349–353.

    Article  CAS  Google Scholar 

  10. van Tol, H., Buzayan, J. M., Feldstein, P. A., Eckstein, F., and Bruening, G. (1990) Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration. Nucleic Acids Res. 18, 1971–1975.

    Article  PubMed  Google Scholar 

  11. Slim, G. and Gait, M. J. (1991) Configurationally defined phosphorotioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 19, 1183–1188.

    Article  PubMed  CAS  Google Scholar 

  12. Saenger, W. (1984) Principles of Nucleic Acid Structure Springer-Verlag, New York, p. 108.

    Google Scholar 

  13. Sawata, S., Shimayama, T., Komiyama, M., Kumar, P. K., Nishikawa, S., and Taira, K. (1993) Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions. Nucleic Acids Res. 21, 5656–60.

    Article  PubMed  CAS  Google Scholar 

  14. Sioud, M. (1994) Interaction between tumour necrosis factor alpha ribozyme and cellular proteins—involvement in ribozyme stability and activity. J. Mol. Biol. 242, 619–629.

    Article  PubMed  CAS  Google Scholar 

  15. Hendry, P., McCall, M. J. Santiago, F. S., and Jennings, P. A. (1995) The in vitro activity of minimised hammerhead ribozymes. Nucleic Acids Res. 23, 3922–3927.

    Article  PubMed  CAS  Google Scholar 

  16. Shimayama, T., Nishikawa, S., and Taira, K. (1995) Extraordinary enhancement of the cleavage activity of a DNA-armed hammerhead ribozyme at elevated concentrations of Mg2+ ions. FEBS Lett. 368, 304–306.

    Article  PubMed  CAS  Google Scholar 

  17. Tsuchihashi, Z., Khosla, M., and Herschlag, D. (1993) Protein enhancement of hammerhead ribozyme catalysis. Science 262, 99–102.

    Article  PubMed  CAS  Google Scholar 

  18. Bertrand, E. L. and Rossit, J. J. (1994) Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1. EMBO J. 13, 2904–2912.

    PubMed  CAS  Google Scholar 

  19. Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R. M. M. (1966) Hydrogen ion buffers for biological research. Biochemistry 5, 467–477.

    Article  PubMed  CAS  Google Scholar 

  20. Veloso, D., Guynn, R. W., Oskarsson, M., and Veech, R. L. (1973) The concentrations of free and bound magnesium in rat tissues. J. Biol. Chem. 248, 4811–4819.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1997 Humana Press Inc.

About this protocol

Cite this protocol

Hendry, P., McCall, M.J., Lockett, T.J. (1997). Defining Optimum Reaction Conditions for Hammerhead Ribozymes. In: Turner, P.C. (eds) Ribozyme Protocols. Methods in Molecular Biology™, vol 74. Humana Press. https://doi.org/10.1385/0-89603-389-9:231

Download citation

  • DOI: https://doi.org/10.1385/0-89603-389-9:231

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-389-4

  • Online ISBN: 978-1-59259-560-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics