Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Dec 15;25(24):5119–5124. doi: 10.1093/nar/25.24.5119

Single substitutions of phosphorothioates in the HDV ribozyme G73 define regions necessary for optimal self-cleaving activity.

N S Prabhu 1, G Dinter-Gottlieb 1, P A Gottlieb 1
PMCID: PMC147158  PMID: 9396824

Abstract

Phosphorothioate (NTPalphaS) analogues were incorporated into the HDV genomic ribozyme by transcription with T7 polymerase. The introduction of a sulfur in place of the pro-Rp oxygen at the phosphate 5'to positions A64, A63, A43, U27, G62, C61, C44, C41, C22and C21appeared to inhibit self-cleavage activity of the G73 genomic ribozyme. Except for position C22, elevated levels of Mg2+rescued the reaction to various extents. When the sites were identified in the RNA sequence, they were clustered in three distinct regions that, in the secondary structure models, are predicted to be primarily single-stranded. Two of these regions have been proposed to form extensive interactions that are thought to involve a homopurine base pair. The third region is thought to be directly associated with assembly of the cleavage site.

Full Text

The Full Text of this article is available as a PDF (143.3 KB).

Selected References

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

  1. Battiste J. L., Tan R., Frankel A. D., Williamson J. R. Binding of an HIV Rev peptide to Rev responsive element RNA induces formation of purine-purine base pairs. Biochemistry. 1994 Mar 15;33(10):2741–2747. doi: 10.1021/bi00176a001. [DOI] [PubMed] [Google Scholar]
  2. Been M. D., Perrotta A. T. Optimal self-cleavage activity of the hepatitis delta virus RNA is dependent on a homopurine base pair in the ribozyme core. RNA. 1995 Dec;1(10):1061–1070. [PMC free article] [PubMed] [Google Scholar]
  3. Belinsky M. G., Britton E., Dinter-Gottlieb G. Modification interference analysis of a self-cleaving RNA from hepatitis delta virus. FASEB J. 1993 Jan;7(1):130–136. doi: 10.1096/fasebj.7.1.8422959. [DOI] [PubMed] [Google Scholar]
  4. Belinsky M., Dinter-Gottlieb G. Self-cleavage of internally deleted hepatitis delta RNAs. Prog Clin Biol Res. 1993;382:89–97. [PubMed] [Google Scholar]
  5. Chowrira B. M., Burke J. M. Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes. Nucleic Acids Res. 1992 Jun 11;20(11):2835–2840. doi: 10.1093/nar/20.11.2835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christian E. L., Yarus M. Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena. J Mol Biol. 1992 Dec 5;228(3):743–758. doi: 10.1016/0022-2836(92)90861-d. [DOI] [PubMed] [Google Scholar]
  7. Christian E. L., Yarus M. Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena. Biochemistry. 1993 May 4;32(17):4475–4480. doi: 10.1021/bi00068a001. [DOI] [PubMed] [Google Scholar]
  8. Eckstein F., Gish G. Phosphorothioates in molecular biology. Trends Biochem Sci. 1989 Mar;14(3):97–100. doi: 10.1016/0968-0004(89)90130-8. [DOI] [PubMed] [Google Scholar]
  9. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Frey P. A., Sammons R. D. Bond order and charge localization in nucleoside phosphorothioates. Science. 1985 May 3;228(4699):541–545. doi: 10.1126/science.2984773. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Heidenreich O., Pieken W., Eckstein F. Chemically modified RNA: approaches and applications. FASEB J. 1993 Jan;7(1):90–96. doi: 10.1096/fasebj.7.1.7678566. [DOI] [PubMed] [Google Scholar]
  14. Jaffe E. K., Cohn M. Divalent cation-dependent stereospecificity of adenosine 5'-O-(2-thiotriphosphate) in the hexokinase and pyruvate kinase reactions. The absolute stereochemistry of the diastereoisomers of adenosine 5'-O-(2-thiotriphosphate). J Biol Chem. 1978 Jul 25;253(14):4823–4825. [PubMed] [Google Scholar]
  15. Jeoung Y. H., Kumar P. K., Suh Y. A., Taira K., Nishikawa S. Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis. Nucleic Acids Res. 1994 Sep 11;22(18):3722–3727. doi: 10.1093/nar/22.18.3722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuo M. Y., Sharmeen L., Dinter-Gottlieb G., Taylor J. Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol. 1988 Dec;62(12):4439–4444. doi: 10.1128/jvi.62.12.4439-4444.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Perrotta A. T., Been M. D. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature. 1991 Apr 4;350(6317):434–436. doi: 10.1038/350434a0. [DOI] [PubMed] [Google Scholar]
  19. Ruffner D. E., Uhlenbeck O. C. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 1990 Oct 25;18(20):6025–6029. doi: 10.1093/nar/18.20.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. SantaLucia J., Jr, Turner D. H. Structure of (rGGCGAGCC)2 in solution from NMR and restrained molecular dynamics. Biochemistry. 1993 Nov 30;32(47):12612–12623. doi: 10.1021/bi00210a009. [DOI] [PubMed] [Google Scholar]
  21. Sharmeen L., Kuo M. Y., Dinter-Gottlieb G., Taylor J. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J Virol. 1988 Aug;62(8):2674–2679. doi: 10.1128/jvi.62.8.2674-2679.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smith J. B., Gottlieb P. A., Dinter-Gottlieb G. A sequence element necessary for self-cleavage of the antigenomic hepatitis delta RNA in 20 M formamide. Biochemistry. 1992 Oct 13;31(40):9629–9635. doi: 10.1021/bi00155a015. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Wu H. N., Lin Y. J., Lin F. P., Makino S., Chang M. F., Lai M. M. Human hepatitis delta virus RNA subfragments contain an autocleavage activity. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1831–1835. doi: 10.1073/pnas.86.6.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu M., Turner D. H. Solution structure of (rGCGGACGC)2 by two-dimensional NMR and the iterative relaxation matrix approach. Biochemistry. 1996 Jul 30;35(30):9677–9689. doi: 10.1021/bi960133q. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES