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. 1999 Sep;5(9):1140–1148. doi: 10.1017/s1355838299990763

Ribozyme cleavage of a 2,5-phosphodiester linkage: mechanism and a restricted divalent metal-ion requirement.

I H Shih 1, M D Been 1
PMCID: PMC1369837  PMID: 10496215

Abstract

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3',5'-phosphodiester linkage at the cleavage site; however, a 2',5'-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2',3'-cyclic phosphate and 5'-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3',5'-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2',5'-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2',5'-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5' to the cleavage site.

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Selected References

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  1. Chen P. J., Kalpana G., Goldberg J., Mason W., Werner B., Gerin J., Taylor J. Structure and replication of the genome of the hepatitis delta virus. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8774–8778. doi: 10.1073/pnas.83.22.8774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chowrira B. M., Burke J. M. Binding and cleavage of nucleic acids by the "hairpin" ribozyme. Biochemistry. 1991 Sep 3;30(35):8518–8522. doi: 10.1021/bi00099a003. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellis K. J., Morrison J. F. Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol. 1982;87:405–426. doi: 10.1016/s0076-6879(82)87025-0. [DOI] [PubMed] [Google Scholar]
  7. Fauzi H., Kawakami J., Nishikawa F., Nishikawa S. Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme. Nucleic Acids Res. 1997 Aug 1;25(15):3124–3130. doi: 10.1093/nar/25.15.3124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Ferré-D'Amaré A. R., Zhou K., Doudna J. A. Crystal structure of a hepatitis delta virus ribozyme. Nature. 1998 Oct 8;395(6702):567–574. doi: 10.1038/26912. [DOI] [PubMed] [Google Scholar]
  10. Herschlag D., Cech T. R. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry. 1990 Nov 6;29(44):10172–10180. doi: 10.1021/bi00496a004. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Moore M. J., Sharp P. A. Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992 May 15;256(5059):992–997. doi: 10.1126/science.1589782. [DOI] [PubMed] [Google Scholar]
  15. Nesbitt S., Hegg L. A., Fedor M. J. An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis. Chem Biol. 1997 Aug;4(8):619–630. doi: 10.1016/s1074-5521(97)90247-7. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Perrotta A. T., Been M. D. Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence. Biochemistry. 1992 Jan 14;31(1):16–21. doi: 10.1021/bi00116a004. [DOI] [PubMed] [Google Scholar]
  18. Perrotta A. T., Been M. D. The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant. Nucleic Acids Res. 1990 Dec 11;18(23):6821–6827. doi: 10.1093/nar/18.23.6821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Piccirilli J. A., Vyle J. S., Caruthers M. H., Cech T. R. Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature. 1993 Jan 7;361(6407):85–88. doi: 10.1038/361085a0. [DOI] [PubMed] [Google Scholar]
  20. Pyle A. M. Ribozymes: a distinct class of metalloenzymes. Science. 1993 Aug 6;261(5122):709–714. doi: 10.1126/science.7688142. [DOI] [PubMed] [Google Scholar]
  21. Rizzetto M., Hoyer B., Canese M. G., Shih J. W., Purcell R. H., Gerin J. L. delta Agent: association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infected chimpanzees. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6124–6128. doi: 10.1073/pnas.77.10.6124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rosenstein S. P., Been M. D. Hepatitis delta virus ribozymes fold to generate a solvent-inaccessible core with essential nucleotides near the cleavage site phosphate. Biochemistry. 1996 Sep 3;35(35):11403–11413. doi: 10.1021/bi9609984. [DOI] [PubMed] [Google Scholar]
  23. Scott E. C., Uhlenbeck O. C. A re-investigation of the thio effect at the hammerhead cleavage site. Nucleic Acids Res. 1999 Jan 15;27(2):479–484. doi: 10.1093/nar/27.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Sharmeen L., Kuo M. Y., Taylor J. Self-ligating RNA sequences on the antigenome of human hepatitis delta virus. J Virol. 1989 Mar;63(3):1428–1430. doi: 10.1128/jvi.63.3.1428-1430.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Suh Y. A., Kumar P. K., Taira K., Nishikawa S. Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions. Nucleic Acids Res. 1993 Jul 11;21(14):3277–3280. doi: 10.1093/nar/21.14.3277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wadkins T. S., Perrotta A. T., Ferré-D'Amaré A. R., Doudna J. A., Been M. D. A nested double pseudoknot is required for self-cleavage activity of both the genomic and antigenomic hepatitis delta virus ribozymes. RNA. 1999 Jun;5(6):720–727. doi: 10.1017/s1355838299990209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wang K. S., Choo Q. L., Weiner A. J., Ou J. H., Najarian R. C., Thayer R. M., Mullenbach G. T., Denniston K. J., Gerin J. L., Houghton M. Structure, sequence and expression of the hepatitis delta (delta) viral genome. Nature. 1986 Oct 9;323(6088):508–514. doi: 10.1038/323508a0. [DOI] [PubMed] [Google Scholar]
  29. Weinstein L. B., Jones B. C., Cosstick R., Cech T. R. A second catalytic metal ion in group I ribozyme. Nature. 1997 Aug 21;388(6644):805–808. doi: 10.1038/42076. [DOI] [PubMed] [Google Scholar]
  30. 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]

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