Skip to main content
NCBI home page
RNA logoLink to RNA
. 2000 Apr;6(4):511–519. doi: 10.1017/s1355838200000042

Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme.

E L Christian 1, N M Kaye 1, M E Harris 1
PMCID: PMC1369932  PMID: 10786842

Abstract

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.

Full Text

The Full Text of this article is available as a PDF (375.0 KB).

Selected References

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

  1. Basu S., Strobel S. A. Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain. RNA. 1999 Nov;5(11):1399–1407. doi: 10.1017/s135583829999115x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Brautigam C. A., Steitz T. A. Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. J Mol Biol. 1998 Mar 27;277(2):363–377. doi: 10.1006/jmbi.1997.1586. [DOI] [PubMed] [Google Scholar]
  4. Burgers P. M., Eckstein F., Hunneman D. H. Stereochemistry of hydrolysis by snake venom phosphodiesterase. J Biol Chem. 1979 Aug 25;254(16):7476–7478. [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Chen J. L., Nolan J. M., Harris M. E., Pace N. R. Comparative photocross-linking analysis of the tertiary structures of Escherichia coli and Bacillus subtilis RNase P RNAs. EMBO J. 1998 Mar 2;17(5):1515–1525. doi: 10.1093/emboj/17.5.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Chen Y., Li X., Gegenheimer P. Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond. Biochemistry. 1997 Mar 4;36(9):2425–2438. doi: 10.1021/bi9620464. [DOI] [PubMed] [Google Scholar]
  11. Christian E. L., Harris M. E. The track of the pre-tRNA 5' leader in the ribonuclease P ribozyme-substrate complex. Biochemistry. 1999 Sep 28;38(39):12629–12638. doi: 10.1021/bi991278a. [DOI] [PubMed] [Google Scholar]
  12. Christian E. L., McPheeters D. S., Harris M. E. Identification of individual nucleotides in the bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-linking. Biochemistry. 1998 Dec 15;37(50):17618–17628. doi: 10.1021/bi982050a. [DOI] [PubMed] [Google Scholar]
  13. Correll C. C., Freeborn B., Moore P. B., Steitz T. A. Metals, motifs, and recognition in the crystal structure of a 5S rRNA domain. Cell. 1997 Nov 28;91(5):705–712. doi: 10.1016/s0092-8674(00)80457-2. [DOI] [PubMed] [Google Scholar]
  14. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Frank D. N., Pace N. R. Ribonuclease P: unity and diversity in a tRNA processing ribozyme. Annu Rev Biochem. 1998;67:153–180. doi: 10.1146/annurev.biochem.67.1.153. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Haas E. S., Brown J. W., Pitulle C., Pace N. R. Further perspective on the catalytic core and secondary structure of ribonuclease P RNA. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2527–2531. doi: 10.1073/pnas.91.7.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Heide C., Pfeiffer T., Nolan J. M., Hartmann R. K. Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA. RNA. 1999 Jan;5(1):102–116. doi: 10.1017/s1355838299981499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kazantsev A. V., Pace N. R. Identification by modification-interference of purine N-7 and ribose 2'-OH groups critical for catalysis by bacterial ribonuclease P. RNA. 1998 Aug;4(8):937–947. doi: 10.1017/s1355838298980384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kirsebom L. A., Svärd S. G. Base pairing between Escherichia coli RNase P RNA and its substrate. EMBO J. 1994 Oct 17;13(20):4870–4876. doi: 10.1002/j.1460-2075.1994.tb06814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Knowles J. R. Enzyme-catalyzed phosphoryl transfer reactions. Annu Rev Biochem. 1980;49:877–919. doi: 10.1146/annurev.bi.49.070180.004305. [DOI] [PubMed] [Google Scholar]
  28. Konforti B. B., Abramovitz D. L., Duarte C. M., Karpeisky A., Beigelman L., Pyle A. M. Ribozyme catalysis from the major groove of group II intron domain 5. Mol Cell. 1998 Feb;1(3):433–441. doi: 10.1016/s1097-2765(00)80043-x. [DOI] [PubMed] [Google Scholar]
  29. Kufel J., Kirsebom L. A. Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6085–6090. doi: 10.1073/pnas.93.12.6085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kufel J., Kirsebom L. A. The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain. RNA. 1998 Jul;4(7):777–788. doi: 10.1017/s1355838298970923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kurz J. C., Niranjanakumari S., Fierke C. A. Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp. Biochemistry. 1998 Feb 24;37(8):2393–2400. doi: 10.1021/bi972530m. [DOI] [PubMed] [Google Scholar]
  32. Loria A., Pan T. Domain structure of the ribozyme from eubacterial ribonuclease P. RNA. 1996 Jun;2(6):551–563. [PMC free article] [PubMed] [Google Scholar]
  33. Loria A., Pan T. Recognition of the 5' leader and the acceptor stem of a pre-tRNA substrate by the ribozyme from Bacillus subtilis RNase P. Biochemistry. 1998 Jul 14;37(28):10126–10133. doi: 10.1021/bi980220d. [DOI] [PubMed] [Google Scholar]
  34. Massire C., Jaeger L., Westhof E. Derivation of the three-dimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis. J Mol Biol. 1998 Jun 19;279(4):773–793. doi: 10.1006/jmbi.1998.1797. [DOI] [PubMed] [Google Scholar]
  35. Michel F., Hanna M., Green R., Bartel D. P., Szostak J. W. The guanosine binding site of the Tetrahymena ribozyme. Nature. 1989 Nov 23;342(6248):391–395. doi: 10.1038/342391a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Narlikar G. J., Herschlag D. Mechanistic aspects of enzymatic catalysis: lessons from comparison of RNA and protein enzymes. Annu Rev Biochem. 1997;66:19–59. doi: 10.1146/annurev.biochem.66.1.19. [DOI] [PubMed] [Google Scholar]
  38. Nolan J. M., Burke D. H., Pace N. R. Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure. Science. 1993 Aug 6;261(5122):762–765. doi: 10.1126/science.7688143. [DOI] [PubMed] [Google Scholar]
  39. Pan T., Fang X., Sosnick T. Pathway modulation, circular permutation and rapid RNA folding under kinetic control. J Mol Biol. 1999 Feb 26;286(3):721–731. doi: 10.1006/jmbi.1998.2516. [DOI] [PubMed] [Google Scholar]
  40. Pan T. Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry. 1995 Jan 24;34(3):902–909. doi: 10.1021/bi00003a024. [DOI] [PubMed] [Google Scholar]
  41. Pan T., Jakacka M. Multiple substrate binding sites in the ribozyme from Bacillus subtilis RNase P. EMBO J. 1996 May 1;15(9):2249–2255. [PMC free article] [PubMed] [Google Scholar]
  42. Pan T., Loria A., Zhong K. Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts between the RNase P RNA and pre-tRNA. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12510–12514. doi: 10.1073/pnas.92.26.12510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pan T., Sosnick T. R. Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity. Nat Struct Biol. 1997 Nov;4(11):931–938. doi: 10.1038/nsb1197-931. [DOI] [PubMed] [Google Scholar]
  44. Patel D. J. Adaptive recognition in RNA complexes with peptides and protein modules. Curr Opin Struct Biol. 1999 Feb;9(1):74–87. doi: 10.1016/s0959-440x(99)80010-4. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Pyle A. M. Role of metal ions in ribozymes. Met Ions Biol Syst. 1996;32:479–520. [PubMed] [Google Scholar]
  47. Reich C., Olsen G. J., Pace B., Pace N. R. Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme. Science. 1988 Jan 8;239(4836):178–181. doi: 10.1126/science.3122322. [DOI] [PubMed] [Google Scholar]
  48. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  49. 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]
  50. Shan S. o., Yoshida A., Sun S., Piccirilli J. A., Herschlag D. Three metal ions at the active site of the Tetrahymena group I ribozyme. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12299–12304. doi: 10.1073/pnas.96.22.12299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Siew D., Zahler N. H., Cassano A. G., Strobel S. A., Harris M. E. Identification of adenosine functional groups involved in substrate binding by the ribonuclease P ribozyme. Biochemistry. 1999 Feb 9;38(6):1873–1883. doi: 10.1021/bi982329r. [DOI] [PubMed] [Google Scholar]
  52. Slim G., Gait M. J. Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 1991 Mar 25;19(6):1183–1188. doi: 10.1093/nar/19.6.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Smith D., Burgin A. B., Haas E. S., Pace N. R. Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis. J Biol Chem. 1992 Feb 5;267(4):2429–2436. [PubMed] [Google Scholar]
  54. 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]
  55. Warnecke J. M., Fürste J. P., Hardt W. D., Erdmann V. A., Hartmann R. K. Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8924–8928. doi: 10.1073/pnas.93.17.8924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weeks K. M., Crothers D. M. Major groove accessibility of RNA. Science. 1993 Sep 17;261(5128):1574–1577. doi: 10.1126/science.7690496. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Yoshida A., Sun S., Piccirilli J. A. A new metal ion interaction in the Tetrahymena ribozyme reaction revealed by double sulfur substitution. Nat Struct Biol. 1999 Apr;6(4):318–321. doi: 10.1038/7551. [DOI] [PubMed] [Google Scholar]
  59. Zarrinkar P. P., Wang J., Williamson J. R. Slow folding kinetics of RNase P RNA. RNA. 1996 Jun;2(6):564–573. [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES