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Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and magnesium binding
Abstract
Mg2+ is important for the RNase activity of the hammerhead ribozyme. To investigate the binding properties of Mg2+ to the hammerhead ribozyme, cleavage rates and CD spectra for substrates containing inosine or guanosine at the cleavage site were measured. The 2-amino group of this guanosine interfered with the rate of the cleavage reaction and did not affect the amount of Mg2+ bound to the hammerhead RNA. The kinetics and CD spectra for chemically synthesized oligoribonucleotides with a Sp or Rp phosphorothioate diester bond at the cleavage site indicated that 1 mol of Mg2+ binds to the pro-R oxygen of phosphate. The binding constant for Mg2+ was about 10(4) M-1, which represents outer-sphere complexation. The hammerhead ribozyme catalyzes the cleavage reaction via an in-line pathway. This mechanism has been proved for RNA cleavage by RNase A by using a modified oligonucleotide that has an Sp phosphorothionate bond at the cleavage site. From these results, we present the reaction pathway and a model for Mg2+ binding to the hammerhead ribozyme.
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Citations
Cited by
Three-dimensional structure of a hammerhead ribozyme
Pley, H. W., Flaherty, K. M., & McKay, D. B. (1994) · Nature · doi:10.1038/372068a0 · Cited by 953
Scott, W. G., Finch, J. T., & Klug, A. (1995) · Cell · doi:10.1016/s0092-8674(05)80004-2 · Cited by 685
Synthesis, deprotection, analysis and purification of RNA and ribosomes
Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., & Usman, N. (1995) · Nucleic Acids Research · doi:10.1093/nar/23.14.2677 · Cited by 398
Metal ion catalysis in the Tetrahymena ribozyme reaction
Piccirilli, J. A., Vyle, J. S., Caruthers, M. H., & Cech, T. R. (1993) · Nature · doi:10.1038/361085a0 · Cited by 385
Noodleman, L., Lovell, T., Han, W.-G., Li, J., & Himo, F. (2004) · Chemical Reviews · doi:10.1021/cr020625a · Cited by 373
Metadata
- Authors
- Koizumi, M., & Ohtsuka, E.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1991-05
- Volume
- 30
- Issue
- 21
- Pages
- 5145-5150
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2059700062
- Cited by
- 181
- References
- 0
- FWCI
- 5.58
- Type
- journal article
- Language
- en
- Open access
- closed
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- Koizumi, M., & Ohtsuka, E. (1991). Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and magnesium binding. Biochemistry. https://doi.org/10.1021/bi00235a005
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