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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
. 1990 Mar;87(5):1668–1672. doi: 10.1073/pnas.87.5.1668

Substrate sequence effects on "hammerhead" RNA catalytic efficiency.

M J Fedor 1, O C Uhlenbeck 1
PMCID: PMC53543  PMID: 1689847

Abstract

The "hammerhead" RNA self-cleaving domain can be assembled from two RNA molecules: a large (approximately 34 nucleotide) ribozyme RNA containing most of the catalytically essential nucleotides and a small (approximately 13 nucleotide) substrate RNA containing the cleavage site. Four such hammerheads that contained identical catalytic core sequences but differed in the base composition of the helices that are involved in substrate binding had been reported to vary in cleavage rates by more than 70-fold under similar reaction conditions. Steady-state kinetic analyses reveal that kcat values are nearly the same for these hammerheads but Km values vary nearly 60-fold. The substrates for reactions having high Km values form aggregates that are virtually nonreactive. These observations demonstrate that the secondary structure of substrate RNA can be a major determinant of hammerhead catalytic efficiency.

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Selected References

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  1. Branch A. D., Robertson H. D., Dickson E. Longer-than-unit-length viroid minus strands are present in RNA from infected plants. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6381–6385. doi: 10.1073/pnas.78.10.6381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buzayan J. M., Gerlach W. L., Bruening G. Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8859–8862. doi: 10.1073/pnas.83.23.8859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cech T. R. The chemistry of self-splicing RNA and RNA enzymes. Science. 1987 Jun 19;236(4808):1532–1539. doi: 10.1126/science.2438771. [DOI] [PubMed] [Google Scholar]
  4. England T. E., Uhlenbeck O. C. Enzymatic oligoribonucleotide synthesis with T4 RNA ligase. Biochemistry. 1978 May 30;17(11):2069–2076. doi: 10.1021/bi00604a008. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Groebe D. R., Uhlenbeck O. C. Characterization of RNA hairpin loop stability. Nucleic Acids Res. 1988 Dec 23;16(24):11725–11735. doi: 10.1093/nar/16.24.11725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 1983 Dec;35(3 Pt 2):849–857. doi: 10.1016/0092-8674(83)90117-4. [DOI] [PubMed] [Google Scholar]
  8. HOFSTEE B. H. J. Specificity of esterases. I. Identification of two pancreatic aliesterases. J Biol Chem. 1952 Nov;199(1):357–364. [PubMed] [Google Scholar]
  9. Hampel A., Tritz R. RNA catalytic properties of the minimum (-)sTRSV sequence. Biochemistry. 1989 Jun 13;28(12):4929–4933. doi: 10.1021/bi00438a002. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Holbrook S. R., Sussman J. L., Kim S. H. Absence of correlation between base-pair sequence and RNA conformation. Science. 1981 Jun 12;212(4500):1275–1277. doi: 10.1126/science.6165084. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Koizumi M., Hayase Y., Iwai S., Kamiya H., Inoue H., Ohtsuka E. Design of RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res. 1989 Sep 12;17(17):7059–7071. doi: 10.1093/nar/17.17.7059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koizumi M., Iwai S., Ohtsuka E. Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett. 1988 Nov 7;239(2):285–288. doi: 10.1016/0014-5793(88)80935-9. [DOI] [PubMed] [Google Scholar]
  15. Kuchino Y., Watanabe S., Harada F., Nishimura S. Primary structure of AUA-specific isoleucine transfer ribonucleic acid from Escherichia coli. Biochemistry. 1980 May 13;19(10):2085–2089. doi: 10.1021/bi00551a013. [DOI] [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. Reich C., Olsen G. J., Pace B., Pace N. R. Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme. Science. 1988 Jan 8;239(4836):178–181. doi: 10.1126/science.3122322. [DOI] [PubMed] [Google Scholar]
  19. Ruffner D. E., Dahm S. C., Uhlenbeck O. C. Studies on the hammerhead RNA self-cleaving domain. Gene. 1989 Oct 15;82(1):31–41. doi: 10.1016/0378-1119(89)90027-9. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  22. Zaug A. J., Grosshans C. A., Cech T. R. Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. Biochemistry. 1988 Dec 13;27(25):8924–8931. doi: 10.1021/bi00425a008. [DOI] [PubMed] [Google Scholar]

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