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. 1998 Jul 15;26(14):3379–3384. doi: 10.1093/nar/26.14.3379

Allosteric regulation of a ribozyme activity through ligand-induced conformational change.

M Araki 1, Y Okuno 1, Y Hara 1, Y Sugiura 1
PMCID: PMC147720  PMID: 9649622

Abstract

An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k cat of Rz3 increases by approximately 10-fold upon addition of saturating amounts of FMN. To determine the rate constants( K m4and k cat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k cat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.

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Selected References

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  1. Breaker R. R., Joyce G. F. Inventing and improving ribozyme function: rational design versus iterative selection methods. Trends Biotechnol. 1994 Jul;12(7):268–275. doi: 10.1016/0167-7799(94)90138-4. [DOI] [PubMed] [Google Scholar]
  2. Cate J. H., Gooding A. R., Podell E., Zhou K., Golden B. L., Kundrot C. E., Cech T. R., Doudna J. A. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science. 1996 Sep 20;273(5282):1678–1685. doi: 10.1126/science.273.5282.1678. [DOI] [PubMed] [Google Scholar]
  3. Chow Christine S., Bogdan Felicia M. A Structural Basis for RNAminus signLigand Interactions. Chem Rev. 1997 Aug 5;97(5):1489–1514. doi: 10.1021/cr960415w. [DOI] [PubMed] [Google Scholar]
  4. Fan P., Suri A. K., Fiala R., Live D., Patel D. J. Molecular recognition in the FMN-RNA aptamer complex. J Mol Biol. 1996 May 10;258(3):480–500. doi: 10.1006/jmbi.1996.0263. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Gerstein M., Lesk A. M., Chothia C. Structural mechanisms for domain movements in proteins. Biochemistry. 1994 Jun 7;33(22):6739–6749. doi: 10.1021/bi00188a001. [DOI] [PubMed] [Google Scholar]
  7. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Jenison R. D., Gill S. C., Pardi A., Polisky B. High-resolution molecular discrimination by RNA. Science. 1994 Mar 11;263(5152):1425–1429. doi: 10.1126/science.7510417. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  12. Patel D. J., Suri A. K., Jiang F., Jiang L., Fan P., Kumar R. A., Nonin S. Structure, recognition and adaptive binding in RNA aptamer complexes. J Mol Biol. 1997 Oct 10;272(5):645–664. doi: 10.1006/jmbi.1997.1281. [DOI] [PubMed] [Google Scholar]
  13. Peracchi A., Beigelman L., Usman N., Herschlag D. Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11522–11527. doi: 10.1073/pnas.93.21.11522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Symons R. H. Small catalytic RNAs. Annu Rev Biochem. 1992;61:641–671. doi: 10.1146/annurev.bi.61.070192.003233. [DOI] [PubMed] [Google Scholar]
  18. Tang J., Breaker R. R. Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection. RNA. 1997 Aug;3(8):914–925. [PMC free article] [PubMed] [Google Scholar]
  19. Tang J., Breaker R. R. Rational design of allosteric ribozymes. Chem Biol. 1997 Jun;4(6):453–459. doi: 10.1016/s1074-5521(97)90197-6. [DOI] [PubMed] [Google Scholar]
  20. Turner D. H., Sugimoto N., Freier S. M. RNA structure prediction. Annu Rev Biophys Biophys Chem. 1988;17:167–192. doi: 10.1146/annurev.bb.17.060188.001123. [DOI] [PubMed] [Google Scholar]
  21. Tuschl T., Eckstein F. Hammerhead ribozymes: importance of stem-loop II for activity. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6991–6994. doi: 10.1073/pnas.90.15.6991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]

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