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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Jul 19;91(15):6977–6981. doi: 10.1073/pnas.91.15.6977

Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions.

D M Long 1, O C Uhlenbeck 1
PMCID: PMC44321  PMID: 7518924

Abstract

A method is described to obtain intramolecular cleavage rates for the hammerhead ribozyme during in vitro transcription. By avoiding RNA purification and renaturation, the potential for formation of inactive conformations of the RNA is minimized. By showing that an intramolecular hammerhead and a closely related intermolecular hammerhead cleave at the same rate under a given set of conditions, we confirm that both reactions probably have the same rate-limiting step. An in vitro selection strategy was used to isolate active hammerheads from a library of molecules where six randomized nucleotides replaced stem-loop II. The sequence and number of nucleotides which replace stem-loop II have large effects on hammerhead cleavage activity. The relative activities of three sequences selected from the intramolecular library are the same when the sequences are transferred into an intermolecular hammerhead background.

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Selected References

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  1. Chamberlin M., Ring J. Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. J Biol Chem. 1973 Mar 25;248(6):2235–2244. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Dichtl B., Pan T., DiRenzo A. B., Uhlenbeck O. C. Replacement of RNA hairpins by in vitro selected tetranucleotides. Nucleic Acids Res. 1993 Feb 11;21(3):531–535. doi: 10.1093/nar/21.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Forster A. C., Davies C., Sheldon C. C., Jeffries A. C., Symons R. H. Self-cleaving viroid and newt RNAs may only be active as dimers. Nature. 1988 Jul 21;334(6179):265–267. doi: 10.1038/334265a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
  9. Forster A. C., Symons R. H. Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell. 1987 Jul 3;50(1):9–16. doi: 10.1016/0092-8674(87)90657-x. [DOI] [PubMed] [Google Scholar]
  10. Goodchild J., Kohli V. Ribozymes that cleave an RNA sequence from human immunodeficiency virus: the effect of flanking sequence on rate. Arch Biochem Biophys. 1991 Feb 1;284(2):386–391. doi: 10.1016/0003-9861(91)90313-8. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Hutchins C. J., Rathjen P. D., Forster A. C., Symons R. H. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 1986 May 12;14(9):3627–3640. doi: 10.1093/nar/14.9.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. SANTER M., AJL S. J. Steroid metabolism by a species of Pseudomonas. II. Direct evidence for the breakdown of testosterone. J Biol Chem. 1952 Nov;199(1):85–89. [PubMed] [Google Scholar]
  20. Sampson J. R., Uhlenbeck O. C. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1033–1037. doi: 10.1073/pnas.85.4.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sheldon C. C., Symons R. H. RNA stem stability in the formation of a self-cleaving hammerhead structure. Nucleic Acids Res. 1989 Jul 25;17(14):5665–5677. doi: 10.1093/nar/17.14.5665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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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