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. 1990 Aug 11;18(15):4447–4451. doi: 10.1093/nar/18.15.4447

Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA.

J M Buzayan 1, H van Tol 1, P A Feldstein 1, G Bruening 1
PMCID: PMC331263  PMID: 1697063

Abstract

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bruening G. Compilation of self-cleaving sequences from plant virus satellite RNAs and other sources. Methods Enzymol. 1989;180:546–558. doi: 10.1016/0076-6879(89)80123-5. [DOI] [PubMed] [Google Scholar]
  2. Buzayan J. M., Feldstein P. A., Bruening G., Eckstein F. RNA mediated formation of a phosphorothioate diester bond. Biochem Biophys Res Commun. 1988 Oct 14;156(1):340–347. doi: 10.1016/s0006-291x(88)80846-5. [DOI] [PubMed] [Google Scholar]
  3. Buzayan J. M., Feldstein P. A., Segrelles C., Bruening G. Autolytic processing of a phosphorothioate diester bond. Nucleic Acids Res. 1988 May 11;16(9):4009–4023. doi: 10.1093/nar/16.9.4009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  6. England T. E., Bruce A. G., Uhlenbeck O. C. Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 1980;65(1):65–74. doi: 10.1016/s0076-6879(80)65011-3. [DOI] [PubMed] [Google Scholar]
  7. Feldstein P. A., Buzayan J. M., van Tol H., deBear J., Gough G. R., Gilham P. T., Bruening G. Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2'-5' phosphodiester. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2623–2627. doi: 10.1073/pnas.87.7.2623. [DOI] [PMC free article] [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. Gish G., Eckstein F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science. 1988 Jun 10;240(4858):1520–1522. doi: 10.1126/science.2453926. [DOI] [PubMed] [Google Scholar]
  10. Griffiths A. D., Potter B. V., Eperon I. C. Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase. Nucleic Acids Res. 1987 May 26;15(10):4145–4162. doi: 10.1093/nar/15.10.4145. [DOI] [PMC free article] [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. 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. Kiefer M. C., Bruening G., Russell M. L. RNA and capsid accumulation in cowpea protoplasts that are resistant to cowpea mosaic virus strain SB. Virology. 1984 Sep;137(2):371–381. doi: 10.1016/0042-6822(84)90229-0. [DOI] [PubMed] [Google Scholar]
  14. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  15. Mei H. Y., Kaaret T. W., Bruice T. C. A computational approach to the mechanism of self-cleavage of hammerhead RNA. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9727–9731. doi: 10.1073/pnas.86.24.9727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pecoraro V. L., Hermes J. D., Cleland W. W. Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP. Biochemistry. 1984 Oct 23;23(22):5262–5271. doi: 10.1021/bi00317a026. [DOI] [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., 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]
  19. Sheldon C. C., Symons R. H. Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 1989 Jul 25;17(14):5679–5685. doi: 10.1093/nar/17.14.5679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Waring R. B. Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution. Nucleic Acids Res. 1989 Dec 25;17(24):10281–10293. doi: 10.1093/nar/17.24.10281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]
  22. van Tol H., Buzayan J. M., Feldstein P. A., Eckstein F., Bruening G. Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration. Nucleic Acids Res. 1990 Apr 25;18(8):1971–1975. doi: 10.1093/nar/18.8.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]

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