Skip to main content
NCBI home page
RNA logoLink to RNA
. 1998 Aug;4(8):937–947. doi: 10.1017/s1355838298980384

Identification by modification-interference of purine N-7 and ribose 2'-OH groups critical for catalysis by bacterial ribonuclease P.

A V Kazantsev 1, N R Pace 1
PMCID: PMC1369671  PMID: 9701285

Abstract

The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. A self-cleaving RNase P RNA-substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 2'-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 2'-hydroxyl that, when substituted with 7-deazaadenine or 2'-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate-phosphate in the tertiary structure of the ribozyme-substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 2'-deoxy-interference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.

Full Text

The Full Text of this article is available as a PDF (576.4 KB).

Selected References

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

  1. Beebe J. A., Kurz J. C., Fierke C. A. Magnesium ions are required by Bacillus subtilis ribonuclease P RNA for both binding and cleaving precursor tRNAAsp. Biochemistry. 1996 Aug 13;35(32):10493–10505. doi: 10.1021/bi960870m. [DOI] [PubMed] [Google Scholar]
  2. Burgin A. B., Pace N. R. Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent. EMBO J. 1990 Dec;9(12):4111–4118. doi: 10.1002/j.1460-2075.1990.tb07633.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cate J. H., Doudna J. A. Metal-binding sites in the major groove of a large ribozyme domain. Structure. 1996 Oct 15;4(10):1221–1229. doi: 10.1016/s0969-2126(96)00129-3. [DOI] [PubMed] [Google Scholar]
  4. Cate J. H., Gooding A. R., Podell E., Zhou K., Golden B. L., Kundrot C. E., Cech T. R., Doudna J. A. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science. 1996 Sep 20;273(5282):1678–1685. doi: 10.1126/science.273.5282.1678. [DOI] [PubMed] [Google Scholar]
  5. Cate J. H., Hanna R. L., Doudna J. A. A magnesium ion core at the heart of a ribozyme domain. Nat Struct Biol. 1997 Jul;4(7):553–558. doi: 10.1038/nsb0797-553. [DOI] [PubMed] [Google Scholar]
  6. Chen J. L., Pace N. R. Identification of the universally conserved core of ribonuclease P RNA. RNA. 1997 Jun;3(6):557–560. [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Conrad F., Hanne A., Gaur R. K., Krupp G. Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. Nucleic Acids Res. 1995 Jun 11;23(11):1845–1853. doi: 10.1093/nar/23.11.1845. [DOI] [PMC free article] [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. Frank D. N., Harris M. E., Pace N. R. Rational design of self-cleaving pre-tRNA-ribonuclease P RNA conjugates. Biochemistry. 1994 Sep 6;33(35):10800–10808. doi: 10.1021/bi00201a030. [DOI] [PubMed] [Google Scholar]
  11. Frank D. N., Pace N. R. In vitro selection for altered divalent metal specificity in the RNase P RNA. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14355–14360. doi: 10.1073/pnas.94.26.14355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gaur R. K., Krupp G. Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme. Nucleic Acids Res. 1993 Jan 11;21(1):21–26. doi: 10.1093/nar/21.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Guerrier-Takada C., Haydock K., Allen L., Altman S. Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli. Biochemistry. 1986 Apr 8;25(7):1509–1515. doi: 10.1021/bi00355a006. [DOI] [PubMed] [Google Scholar]
  17. Hardt W. D., Erdmann V. A., Hartmann R. K. Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding. RNA. 1996 Dec;2(12):1189–1198. [PMC free article] [PubMed] [Google Scholar]
  18. Hardt W. D., Warnecke J. M., Erdmann V. A., Hartmann R. K. Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding. EMBO J. 1995 Jun 15;14(12):2935–2944. doi: 10.1002/j.1460-2075.1995.tb07293.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harris M. E., Kazantsev A. V., Chen J. L., Pace N. R. Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking. RNA. 1997 Jun;3(6):561–576. [PMC free article] [PubMed] [Google Scholar]
  20. Harris M. E., Nolan J. M., Malhotra A., Brown J. W., Harvey S. C., Pace N. R. Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA. EMBO J. 1994 Sep 1;13(17):3953–3963. doi: 10.1002/j.1460-2075.1994.tb06711.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Harris M. E., Pace N. R. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA. 1995 Apr;1(2):210–218. [PMC free article] [PubMed] [Google Scholar]
  22. Jack A., Ladner J. E., Rhodes D., Brown R. S., Klug A. A crystallographic study of metal-binding to yeast phenylalanine transfer RNA. J Mol Biol. 1977 Apr 15;111(3):315–328. doi: 10.1016/s0022-2836(77)80054-5. [DOI] [PubMed] [Google Scholar]
  23. Jucker F. M., Pardi A. GNRA tetraloops make a U-turn. RNA. 1995 Apr;1(2):219–222. [PMC free article] [PubMed] [Google Scholar]
  24. Pace N. R., Brown J. W. Evolutionary perspective on the structure and function of ribonuclease P, a ribozyme. J Bacteriol. 1995 Apr;177(8):1919–1928. doi: 10.1128/jb.177.8.1919-1928.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Quigley G. J., Rich A. Structural domains of transfer RNA molecules. Science. 1976 Nov 19;194(4267):796–806. doi: 10.1126/science.790568. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Smith D., Pace N. R. Multiple magnesium ions in the ribonuclease P reaction mechanism. Biochemistry. 1993 May 25;32(20):5273–5281. doi: 10.1021/bi00071a001. [DOI] [PubMed] [Google Scholar]
  28. Strobel S. A., Shetty K. Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2903–2908. doi: 10.1073/pnas.94.7.2903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Westhof E., Altman S. Three-dimensional working model of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5133–5137. doi: 10.1073/pnas.91.11.5133. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES