Skip to main content
NCBI home page
RNA logoLink to RNA
. 2002 Jul;8(7):933–947. doi: 10.1017/s1355838202025025

Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P.

Sharon M Crary 1, Jeffrey C Kurz 1, Carol A Fierke 1
PMCID: PMC1370310  PMID: 12166648

Abstract

Ribonuclease P (RNase P) is a ribonucleoprotein that requires magnesium ions to catalyze the 5' maturation of transfer RNA. To identify interactions essential for catalysis, the properties of RNase P containing single sulfur substitutions for nonbridging phosphodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic experiments. Sulfur substitution at the nonbridging oxygens of the phosphodiester bond of nucleotide U51 only modestly affects catalysis. However, phosphorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300-4,000-fold for P RNA and 500-15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of pre-tRNA(Asp), highlighting the importance of this region for catalysis. Furthermore, addition of manganese enhances pre-tRNA cleavage catalyzed by B. subtilis RNase P RNA containing an Sp phosphorothioate modification at A49, as observed for Escherichia coli P RNA [Christian et al., RNA, 2000, 6:511-519], suggesting that an essential metal ion may be coordinated at this site. In contrast, no manganese rescue is observed for the A49 Sp phosphorothioate modification in RNase P holoenzyme. These differential manganese rescue effects, along with affinity cleavage, suggest that the protein component may interact with a metal ion bound near A49 in helix P4 of P RNA.

Full Text

The Full Text of this article is available as a PDF (266.7 KB).

Selected References

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

  1. Anderson C. F., Record M. T., Jr Salt-nucleic acid interactions. Annu Rev Phys Chem. 1995;46:657–700. doi: 10.1146/annurev.pc.46.100195.003301. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Beebe J. A., Fierke C. A. A kinetic mechanism for cleavage of precursor tRNA(Asp) catalyzed by the RNA component of Bacillus subtilis ribonuclease P. Biochemistry. 1994 Aug 30;33(34):10294–10304. doi: 10.1021/bi00200a009. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Biswas R., Ledman D. W., Fox R. O., Altman S., Gopalan V. Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe. J Mol Biol. 2000 Feb 11;296(1):19–31. doi: 10.1006/jmbi.1999.3443. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Burgers P. M., Eckstein F. Absolute configuration of the diastereomers of adenosine 5'-O-(1-thiotriphosphate): consequences for the stereochemistry of polymerization by DNA-dependent RNA polymerase from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4798–4800. doi: 10.1073/pnas.75.10.4798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Christian E. L., Kaye N. M., Harris M. E. Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme. RNA. 2000 Apr;6(4):511–519. doi: 10.1017/s1355838200000042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Christian E. L., Kaye N. M., Harris M. E. Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme. RNA. 2000 Apr;6(4):511–519. doi: 10.1017/s1355838200000042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Crary S. M., Niranjanakumari S., Fierke C. A. The protein component of Bacillus subtilis ribonuclease P increases catalytic efficiency by enhancing interactions with the 5' leader sequence of pre-tRNAAsp. Biochemistry. 1998 Jun 30;37(26):9409–9416. doi: 10.1021/bi980613c. [DOI] [PubMed] [Google Scholar]
  17. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  18. Fierke C. A., Hammes G. G. Transient kinetic approaches to enzyme mechanisms. Methods Enzymol. 1995;249:3–37. doi: 10.1016/0076-6879(95)49029-9. [DOI] [PubMed] [Google Scholar]
  19. Frank D. N., Adamidi C., Ehringer M. A., Pitulle C., Pace N. R. Phylogenetic-comparative analysis of the eukaryal ribonuclease P RNA. RNA. 2000 Dec;6(12):1895–1904. doi: 10.1017/s1355838200001461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Frank D. N., Ellington A. E., Pace N. R. In vitro selection of RNase P RNA reveals optimized catalytic activity in a highly conserved structural domain. RNA. 1996 Dec;2(12):1179–1188. [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Gardiner K. J., Marsh T. L., Pace N. R. Ion dependence of the Bacillus subtilis RNase P reaction. J Biol Chem. 1985 May 10;260(9):5415–5419. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Horton T. E., Maderia M., DeRose V. J. Impact of phosphorothioate substitutions on the thermodynamic stability of an RNA GAAA tetraloop: an unexpected stabilization. Biochemistry. 2000 Jul 18;39(28):8201–8207. doi: 10.1021/bi000141d. [DOI] [PubMed] [Google Scholar]
  28. Hunsicker L. M., DeRose V. J. Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes. J Inorg Biochem. 2000 Jul 1;80(3-4):271–281. doi: 10.1016/s0162-0134(00)00079-9. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Kirsebom L. A. RNase P--a 'Scarlet Pimpernel'. Mol Microbiol. 1995 Aug;17(3):411–420. doi: 10.1111/j.1365-2958.1995.mmi_17030411.x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Kurz J. C., Fierke C. A. Ribonuclease P: a ribonucleoprotein enzyme. Curr Opin Chem Biol. 2000 Oct;4(5):553–558. doi: 10.1016/s1367-5931(00)00131-9. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Manning G. S., Ray J. Counterion condensation revisited. J Biomol Struct Dyn. 1998 Oct;16(2):461–476. doi: 10.1080/07391102.1998.10508261. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  37. Misra V. K., Draper D. E. On the role of magnesium ions in RNA stability. Biopolymers. 1998;48(2-3):113–135. doi: 10.1002/(SICI)1097-0282(1998)48:2<113::AID-BIP3>3.0.CO;2-Y. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Niranjanakumari S., Kurz J. C., Fierke C. A. Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis. Nucleic Acids Res. 1998 Jul 1;26(13):3090–3096. doi: 10.1093/nar/26.13.3090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Niranjanakumari S., Stams T., Crary S. M., Christianson D. W., Fierke C. A. Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15212–15217. doi: 10.1073/pnas.95.26.15212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. 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]
  45. 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]
  46. Perrotta A. T., Been M. D. A toggle duplex in hepatitis delta virus self-cleaving RNA that stabilizes an inactive and a salt-dependent pro-active ribozyme conformation. J Mol Biol. 1998 Jun 5;279(2):361–373. doi: 10.1006/jmbi.1998.1798. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Schatz D., Leberman R., Eckstein F. Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6132–6136. doi: 10.1073/pnas.88.14.6132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schmitz M., Tinoco I., Jr Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA. RNA. 2000 Sep;6(9):1212–1225. doi: 10.1017/s1355838200000881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shan S. O., Herschlag D. An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction. RNA. 2000 Jun;6(6):795–813. doi: 10.1017/s1355838200000649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Shi H., Moore P. B. The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited. RNA. 2000 Aug;6(8):1091–1105. doi: 10.1017/s1355838200000364. [DOI] [PMC free article] [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. Smith J. S., Nikonowicz E. P. Phosphorothioate substitution can substantially alter RNA conformation. Biochemistry. 2000 May 16;39(19):5642–5652. doi: 10.1021/bi992712b. [DOI] [PubMed] [Google Scholar]
  56. Suzumura K., Warashina M., Yoshinari K., Tanaka Y., Kuwabara T., Orita M., Taira K. Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme. FEBS Lett. 2000 May 4;473(1):106–112. doi: 10.1016/s0014-5793(00)01499-x. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Warnecke J. M., Held R., Busch S., Hartmann R. K. Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis. J Mol Biol. 1999 Jul 9;290(2):433–445. doi: 10.1006/jmbi.1999.2890. [DOI] [PubMed] [Google Scholar]
  59. Warnecke J. M., Sontheimer E. J., Piccirilli J. A., Hartmann R. K. Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage. Nucleic Acids Res. 2000 Feb 1;28(3):720–727. doi: 10.1093/nar/28.3.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Waugh D. S., Pace N. R. Gap-scan deletion analysis of Bacillus subtilis RNase P RNA. FASEB J. 1993 Jan;7(1):188–195. doi: 10.1096/fasebj.7.1.7678561. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. 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]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES