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. 1997 Jun 1;25(11):2189–2196. doi: 10.1093/nar/25.11.2189

Differences in the phosphate oxygen requirements for self-cleavage by the extended and prototypical hammerhead forms.

O Mitrasinovic 1, L M Epstein 1
PMCID: PMC146715  PMID: 9153320

Abstract

The hammerhead self-cleaving motif occurs in a variety of RNAs that infect plants and consists of three non-conserved helices connected by a highly conserved central core. A variant hammerhead, called the extended hammerhead, is found in satellite 2 transcripts from a variety of caudate amphibians. The extended hammerhead has the same core as the prototypical hammerhead, but has unusually conserved sequence and structural elements in its peripheral helices. Here we present the results of a thiophosphate substitution interference analysis of the pro-Rp phosphate oxygen requirements in the two hammerhead forms. Five pro-Rp phosphate oxygens, all in the central core, were found to be important for self-cleavage by the prototypical hammerhead. A similar set of core positions were important for self-cleavage by the extended hammerhead, but five non-core positions were also found to be important. Thiosubstitution at one of these positions had the most severe effect on self-cleavage observed in this analysis. Mn2+ did not alleviate this negative effect, indicating that this position was not part of a divalent cation binding site. We propose that novel tertiary interactions in the extended hammerhead help form the same catalytic core structure as that used by the prototypical plant virus hammerhead.

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Selected References

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  1. Been M. D., Perrotta A. T., Rosenstein S. P. Secondary structure of the self-cleaving RNA of hepatitis delta virus: applications to catalytic RNA design. Biochemistry. 1992 Dec 1;31(47):11843–11852. doi: 10.1021/bi00162a024. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Buzayan J. M., van Tol H., Feldstein P. A., Bruening G. Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA. Nucleic Acids Res. 1990 Aug 11;18(15):4447–4451. doi: 10.1093/nar/18.15.4447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Coats S. R., Zhang Y., Epstein L. M. Transcription of satellite 2 DNA from the newt is driven by a snRNA type of promoter. Nucleic Acids Res. 1994 Nov 11;22(22):4697–4704. doi: 10.1093/nar/22.22.4697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cremisi F., Scarabino D., Carluccio M. A., Salvadori P., Barsacchi G. A newt ribozyme: a catalytic activity in search of a function. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1651–1655. doi: 10.1073/pnas.89.5.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  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. Epstein L. M., Coats S. R. Tissue-specific permutations of self-cleaving newt satellite-2 transcripts. Gene. 1991 Nov 15;107(2):213–218. doi: 10.1016/0378-1119(91)90321-2. [DOI] [PubMed] [Google Scholar]
  11. Epstein L. M., Gall J. G. Self-cleaving transcripts of satellite DNA from the newt. Cell. 1987 Feb 13;48(3):535–543. doi: 10.1016/0092-8674(87)90204-2. [DOI] [PubMed] [Google Scholar]
  12. Epstein L. M., Mahon K. A., Gall J. G. Transcription of a satellite DNA in the newt. J Cell Biol. 1986 Oct;103(4):1137–1144. doi: 10.1083/jcb.103.4.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fedor M. J., Uhlenbeck O. C. Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry. 1992 Dec 8;31(48):12042–12054. doi: 10.1021/bi00163a012. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Fu D. J., Rajur S. B., McLaughlin L. W. Importance of specific guanosine N7-nitrogens and purine amino groups for efficient cleavage by a hammerhead ribozyme. Biochemistry. 1993 Oct 12;32(40):10629–10637. doi: 10.1021/bi00091a013. [DOI] [PubMed] [Google Scholar]
  17. Garrett T. A., Pabón-Peña L. M., Gokaldas N., Epstein L. M. Novel requirements in peripheral structures of the extended satellite 2 hammerhead. RNA. 1996 Jul;2(7):699–706. [PMC free article] [PubMed] [Google Scholar]
  18. Green B., Pabón-Peña L. M., Graham T. A., Peach S. E., Coats S. R., Epstein L. M. Conserved sequence and functional domains in satellite 2 from three families of salamanders. Mol Biol Evol. 1993 Jul;10(4):732–750. doi: 10.1093/oxfordjournals.molbev.a040041. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Hendry P., Moghaddam M. J., McCall M. J., Jennings P. A., Ebel S., Brown T. Using linkers to investigate the spatial separation of the conserved nucleotides A9 and G12 in the hammerhead ribozyme. Biochim Biophys Acta. 1994 Oct 18;1219(2):405–412. doi: 10.1016/0167-4781(94)90065-5. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Hertel K. J., Pardi A., Uhlenbeck O. C., Koizumi M., Ohtsuka E., Uesugi S., Cedergren R., Eckstein F., Gerlach W. L., Hodgson R. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. doi: 10.1093/nar/20.12.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Heus H. A., Uhlenbeck O. C., Pardi A. Sequence-dependent structural variations of hammerhead RNA enzymes. Nucleic Acids Res. 1990 Mar 11;18(5):1103–1108. doi: 10.1093/nar/18.5.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jaffe E. K., Cohn M. Diastereomers of the nucleoside phosphorothioates as probes of the structure of the metal nucleotide substrates and of the nucleotide binding site of yeast hexokinase. J Biol Chem. 1979 Nov 10;254(21):10839–10845. [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Ng M. M., Benseler F., Tuschl T., Eckstein F. Isoguanosine substitution of conserved adenosines in the hammerhead ribozyme. Biochemistry. 1994 Oct 11;33(40):12119–12126. doi: 10.1021/bi00206a015. [DOI] [PubMed] [Google Scholar]
  31. Pabón-Peña L. M., Zhang Y., Epstein L. M. Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure. Mol Cell Biol. 1991 Dec;11(12):6109–6115. doi: 10.1128/mcb.11.12.6109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pley H. W., Flaherty K. M., McKay D. B. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994 Nov 3;372(6501):68–74. doi: 10.1038/372068a0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Scott W. G., Finch J. T., Klug A. The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell. 1995 Jun 30;81(7):991–1002. doi: 10.1016/s0092-8674(05)80004-2. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Tuschl T., Gohlke C., Jovin T. M., Westhof E., Eckstein F. A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements. Science. 1994 Nov 4;266(5186):785–789. doi: 10.1126/science.7973630. [DOI] [PubMed] [Google Scholar]
  38. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  39. Uhlenbeck O. C. Hammerhead: Part 2. Nat Struct Biol. 1995 Aug;2(8):610–614. doi: 10.1038/nsb0895-610. [DOI] [PubMed] [Google Scholar]
  40. Williams D. M., Pieken W. A., Eckstein F. Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):918–921. doi: 10.1073/pnas.89.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yang J. H., Usman N., Chartrand P., Cedergren R. Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. Biochemistry. 1992 Jun 2;31(21):5005–5009. doi: 10.1021/bi00136a013. [DOI] [PubMed] [Google Scholar]
  42. Zhang Y., Epstein L. M. Cloning and characterization of extended hammerheads from a diverse set of caudate amphibians. Gene. 1996 Jun 26;172(2):183–190. doi: 10.1016/0378-1119(96)00126-6. [DOI] [PubMed] [Google Scholar]

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