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. 2001 Feb;7(2):233–241. doi: 10.1017/s1355838201001352

The Bacillus subtilis RNase P holoenzyme contains two RNase P RNA and two RNase P protein subunits.

X W Fang 1, X J Yang 1, K Littrell 1, S Niranjanakumari 1, P Thiyagarajan 1, C A Fierke 1, T R Sosnick 1, T Pan 1
PMCID: PMC1370081  PMID: 11233980

Abstract

Ribonuclease P (RNase P) catalyzes the 5' maturation of precursor tRNA transcripts and, in bacteria, is composed of a catalytic RNA and a protein. We investigated the oligomerization state and the shape of the RNA alone and the holoenzyme of Bacillus subtilis RNase P in the absence of substrate by synchrotron small-angle X-ray scattering and affinity retention. The B. subtilis RNase P RNA alone is a monomer; however, the scattering profile changes upon the addition of monovalent ions, possibly suggesting different interdomain angles. To our surprise, the X-ray scattering data combined with the affinity retention results indicate that the holoenzyme contains two RNase P RNA and two RNase P protein molecules. We propose a structural model of the holoenzyme with a symmetrical arrangement of the two RNA subunits, consistent with the X-ray scattering results. This (P RNA)2(P protein)2 complex likely binds substrate differently than the conventional (P RNA)1(P protein)1 complex; therefore, the function of the B. subtilis RNase P holoenzyme may be more diverse than previously thought. These revisions to our knowledge of the RNase P holoenzyme suggest a more versatile role for proteins in ribonucleoprotein complexes.

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Selected References

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  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. 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]
  3. 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]
  4. England T. E., Bruce A. G., Uhlenbeck O. C. Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 1980;65(1):65–74. doi: 10.1016/s0076-6879(80)65011-3. [DOI] [PubMed] [Google Scholar]
  5. Fang X., Littrell K., Yang X. J., Henderson S. J., Siefert S., Thiyagarajan P., Pan T., Sosnick T. R. Mg2+-dependent compaction and folding of yeast tRNAPhe and the catalytic domain of the B. subtilis RNase P RNA determined by small-angle X-ray scattering. Biochemistry. 2000 Sep 12;39(36):11107–11113. doi: 10.1021/bi000724n. [DOI] [PubMed] [Google Scholar]
  6. Fang X., Pan T., Sosnick T. R. A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme. Biochemistry. 1999 Dec 21;38(51):16840–16846. doi: 10.1021/bi991700n. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Gopalan V., Golbik R., Schreiber G., Fersht A. R., Altman S. Fluorescence properties of a tryptophan residue in an aromatic core of the protein subunit of ribonuclease P from Escherichia coli. J Mol Biol. 1997 Apr 11;267(4):765–769. doi: 10.1006/jmbi.1997.0907. [DOI] [PubMed] [Google Scholar]
  10. Gopalan V., Kühne H., Biswas R., Li H., Brudvig G. W., Altman S. Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using electron paramagnetic resonance spectroscopy. Biochemistry. 1999 Feb 9;38(6):1705–1714. doi: 10.1021/bi9807106. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Guerrier-Takada C., McClain W. H., Altman S. Cleavage of tRNA precursors by the RNA subunit of E. coli ribonuclease P (M1 RNA) is influenced by 3'-proximal CCA in the substrates. Cell. 1984 Aug;38(1):219–224. doi: 10.1016/0092-8674(84)90543-9. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Liu F., Altman S. Differential evolution of substrates for an RNA enzyme in the presence and absence of its protein cofactor. Cell. 1994 Jul 1;77(7):1093–1100. doi: 10.1016/0092-8674(94)90448-0. [DOI] [PubMed] [Google Scholar]
  16. Loria A., Niranjanakumari S., Fierke C. A., Pan T. Recognition of a pre-tRNA substrate by the Bacillus subtilis RNase P holoenzyme. Biochemistry. 1998 Nov 3;37(44):15466–15473. doi: 10.1021/bi9816507. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Loria A., Pan T. The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site. Biochemistry. 1999 Jul 6;38(27):8612–8620. doi: 10.1021/bi990691f. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. McClain W. H., Guerrier-Takada C., Altman S. Model substrates for an RNA enzyme. Science. 1987 Oct 23;238(4826):527–530. doi: 10.1126/science.2443980. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Odell L., Huang V., Jakacka M., Pan T. Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P. Nucleic Acids Res. 1998 Aug 15;26(16):3717–3723. doi: 10.1093/nar/26.16.3717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Stams T., Niranjanakumari S., Fierke C. A., Christianson D. W. Ribonuclease P protein structure: evolutionary origins in the translational apparatus. Science. 1998 May 1;280(5364):752–755. doi: 10.1126/science.280.5364.752. [DOI] [PubMed] [Google Scholar]
  27. Svärd S. G., Kirsebom L. A. Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site. J Mol Biol. 1992 Oct 20;227(4):1019–1031. doi: 10.1016/0022-2836(92)90518-o. [DOI] [PubMed] [Google Scholar]
  28. Talbot S. J., Altman S. Gel retardation analysis of the interaction between C5 protein and M1 RNA in the formation of the ribonuclease P holoenzyme from Escherichia coli. Biochemistry. 1994 Feb 15;33(6):1399–1405. doi: 10.1021/bi00172a016. [DOI] [PubMed] [Google Scholar]
  29. Talbot S. J., Altman S. Kinetic and thermodynamic analysis of RNA-protein interactions in the RNase P holoenzyme from Escherichia coli. Biochemistry. 1994 Feb 15;33(6):1406–1411. doi: 10.1021/bi00172a017. [DOI] [PubMed] [Google Scholar]
  30. Tallsjö A., Kirsebom L. A. Product release is a rate-limiting step during cleavage by the catalytic RNA subunit of Escherichia coli RNase P. Nucleic Acids Res. 1993 Jan 11;21(1):51–57. doi: 10.1093/nar/21.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thiyagarajan P., Henderson S. J., Joachimiak A. Solution structures of GroEL and its complex with rhodanese from small-angle neutron scattering. Structure. 1996 Jan 15;4(1):79–88. doi: 10.1016/s0969-2126(96)00011-1. [DOI] [PubMed] [Google Scholar]
  32. Tranguch A. J., Engelke D. R. Comparative structural analysis of nuclear RNase P RNAs from yeast. J Biol Chem. 1993 Jul 5;268(19):14045–14055. [PubMed] [Google Scholar]
  33. Vioque A., Arnez J., Altman S. Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli. J Mol Biol. 1988 Aug 20;202(4):835–848. doi: 10.1016/0022-2836(88)90562-1. [DOI] [PubMed] [Google Scholar]

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