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Structural perspective on the activation of RNase P RNA by protein

Nature Structural & Molecular Biology volume 12, pages 958–964 (2005)Cite this article

Abstract

Ribonucleoprotein particles are central to numerous cellular pathways, but their study in vitro is often complicated by heterogeneity and aggregation. We describe a new technique to characterize these complexes trapped as homogeneous species in a nondenaturing gel. Using this technique, in conjunction with phosphorothioate footprinting analysis, we identify the protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a protein cofactor to catalyze the 5′ maturation of precursor transfer RNA (pre-tRNA). Our results show that the protein binds to a patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the protein in substrate recognition and support a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.

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Figure 1: Nondenaturing gel shift and phosphorothioate-iodine protection of RNase P RNA by RNase P protein.
Figure 2: Increased phosphorothioate-iodine cleavage of residues in B. stearothermophilus RNA in the presence of proteins.
Figure 3: Phosphorothioate-iodine data mapped onto the secondary structures of B. stearothermophilus and E. coli RNAs.
Figure 4: Protection of RNase P RNA by RNase P protein.
Figure 5: Ternary complex model.
Figure 6: Folding transition of the B. stearothermophilus RNA.

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References

  1. 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 35, 849–857 (1983).

    CAS  PubMed  Google Scholar 

  2. Reich, C., Olsen, G.J., Pace, B. & Pace, N.R. Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme. Science 239, 178–181 (1988).

    CAS  PubMed  Google Scholar 

  3. Kurz, J.C., Niranjanakumari, S. & Fierke, C.A. Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp. Biochemistry 37, 2393–2400 (1998).

    CAS  PubMed  Google Scholar 

  4. Buck, A.H., Dalby, A.B., Poole, A., Kazantsev, A.V. & Pace, N.R. Protein activation of a ribozyme: the role of bacterial RNase P protein. EMBO J. published online 15 September 2005 (10.1038/sj.emboj.7600805).

  5. Kazantsev, A.V. et al. Crystal structure of a bacterial ribonuclease P RNA. Proc. Natl. Acad. Sci. USA 102, 13392–13397 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Loria, A., Niranjanakumari, S., Fierke, C.A. & Pan, T. Recognition of a pre-tRNA substrate by the Bacillus subtilis RNase P holoenzyme. Biochemistry 37, 15466–15473 (1998).

    CAS  PubMed  Google Scholar 

  7. Rox, C., Feltens, R., Pfeiffer, T. & Hartmann, R.K. Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme. J. Mol. Biol. 315, 551–560 (2002).

    CAS  PubMed  Google Scholar 

  8. Day-Storms, J.J., Niranjanakumari, S. & Fierke, C.A. Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay. RNA 10, 1595–1608 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Loria, A. & Pan, T. Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein. Nucleic Acids Res. 29, 1892–1897 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Waugh, D.S. & Pace, N.R. Complementation of an RNase P RNA (rnpB) gene deletion in Escherichia coli by homologous genes from distantly related eubacteria. J. Bacteriol. 172, 6316–6322 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Vioque, A., Arnez, J. & Altman, S. Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli. J. Mol. Biol. 202, 835–848 (1988).

    CAS  PubMed  Google Scholar 

  12. 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 33, 1399–1405 (1994).

    CAS  PubMed  Google Scholar 

  13. Westhof, E., Wesolowski, D. & Altman, S. Mapping in three dimensions of regions in a catalytic RNA protected from attack by an Fe(II)-EDTA reagent. J. Mol. Biol. 258, 600–613 (1996).

    CAS  PubMed  Google Scholar 

  14. Sharkady, S.M. & Nolan, J.M. Bacterial ribonuclease P holoenzyme crosslinking analysis reveals protein interaction sites on the RNA subunit. Nucleic Acids Res. 29, 3848–3856 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 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. 296, 19–31 (2000).

    CAS  PubMed  Google Scholar 

  16. Tsai, H.Y., Masquida, B., Biswas, R., Westhof, E. & Gopalan, V. Molecular modeling of the three-dimensional structure of the bacterial RNase P holoenzyme. J. Mol. Biol. 325, 661–675 (2003).

    CAS  PubMed  Google Scholar 

  17. Fang, X.W. et al. The Bacillus subtilis RNase P holoenzyme contains two RNase P RNA and two RNase P protein subunits. RNA 7, 233–241 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Haas, E.S., Banta, A.B., Harris, J.K., Pace, N.R. & Brown, J.W. Structure and evolution of ribonuclease P RNA in Gram-positive bacteria. Nucleic Acids Res. 24, 4775–4782 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Brown, J.W. & Pace, N.R. Ribonuclease P RNA and protein subunits from bacteria. Nucleic Acids Res. 20, 1451–1456 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kazantsev, A.V. et al. High-resolution structure of RNase P protein from Thermotoga maritima. Proc. Natl. Acad. Sci. USA 100, 7497–7502 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Stams, T., Niranjanakumari, S., Fierke, C.A. & Christianson, D.W. Ribonuclease P protein structure: evolutionary origins in the translational apparatus. Science 280, 752–755 (1998).

    CAS  PubMed  Google Scholar 

  22. Gish, G. & Eckstein, F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520–1522 (1988).

    CAS  PubMed  Google Scholar 

  23. Winkler, W.C., Nahvi, A., Roth, A., Collins, J.A. & Breaker, R.R. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286 (2004).

    CAS  PubMed  Google Scholar 

  24. Christian, E.L., Zahler, N.H., Kaye, N.M. & Harris, M.E. Analysis of substrate recognition by the ribonucleoprotein endonuclease RNase P. Methods 28, 307–322 (2002).

    CAS  PubMed  Google Scholar 

  25. 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. 17, 1515–1525 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Kufel, J. & Kirsebom, L.A. The P15-loop of Escherichia coli RNase P RNA is an autonomous divalent metal ion binding domain. RNA 4, 777–788 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Oh, B.K., Frank, D.N. & Pace, N.R. Participation of the 3′-CCA of tRNA in the binding of catalytic Mg2+ ions by ribonuclease P. Biochemistry 37, 7277–7283 (1998).

    CAS  PubMed  Google Scholar 

  28. Kazakov, S. & Altman, S. Site-specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. Proc. Natl. Acad. Sci. USA 88, 9193–9197 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Massire, C., Jaeger, L. & Westhof, E. Derivation of the three-dimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis. J. Mol. Biol. 279, 773–793 (1998).

    CAS  PubMed  Google Scholar 

  30. 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. USA 95, 15212–15217 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 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 37, 9409–9416 (1998).

    CAS  PubMed  Google Scholar 

  32. 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. 4, 931–938 (1997).

    CAS  PubMed  Google Scholar 

  33. Fang, X., Pan, T. & Sosnick, T.R. A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme. Biochemistry 38, 16840–16846 (1999).

    CAS  PubMed  Google Scholar 

  34. Haas, E.S., Morse, D.P., Brown, J.W., Schmidt, F.J. & Pace, N.R. Long-range structure in ribonuclease P RNA. Science 254, 853–856 (1991).

    CAS  PubMed  Google Scholar 

  35. Beebe, J.A. & Fierke, C.A. A kinetic mechanism for cleavage of precursor tRNAAsp catalyzed by the RNA component of Bacillus subtilis ribonuclease P. Biochemistry 33, 10294–10304 (1994).

    CAS  PubMed  Google Scholar 

  36. Smith, D. & Pace, N.R. Multiple magnesium ions in the ribonuclease P reaction mechanism. Biochemistry 32, 5273–5281 (1993).

    CAS  PubMed  Google Scholar 

  37. Talbot, S.J. & Altman, S. Kinetic and thermodynamic analysis of RNA-protein interactions in the RNase P holoenzyme from Escherichia coli. Biochemistry 33, 1406–1411 (1994).

    CAS  PubMed  Google Scholar 

  38. Williamson, J.R. Induced fit in RNA-protein recognition. Nat. Struct. Biol. 7, 834–837 (2000).

    CAS  PubMed  Google Scholar 

  39. Kurz, J.C. & Fierke, C.A. The affinity of magnesium binding sites in the Bacillus subtilis RNase P × pre-tRNA complex is enhanced by the protein subunit. Biochemistry 41, 9545–9558 (2002).

    CAS  PubMed  Google Scholar 

  40. Frank, D.N. & Pace, N.R. In vitro selection for altered divalent metal specificity in the RNase P RNA. Proc. Natl. Acad. Sci. USA 94, 14355–14360 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 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 6, 511–519 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Crary, S.M., Kurz, J.C. & Fierke, C.A. Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P. RNA 8, 933–947 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Brannvall, M., Fredrik Pettersson, B.M. & Kirsebom, L.A. The residue immediately upstream of the RNase P cleavage site is a positive determinant. Biochimie 84, 693–703 (2002).

    PubMed  Google Scholar 

  44. LaGrandeur, T.E., Huttenhofer, A., Noller, H.F. & Pace, N.R. Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA. EMBO J. 13, 3945–3952 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Zahler, N.H., Christian, E.L. & Harris, M.E. Recognition of the 5′ leader of pre-tRNA substrates by the active site of ribonuclease P. RNA 9, 734–745 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Zuleeg, T. et al. Correlation between processing efficiency for ribonuclease P minimal substrates and conformation of the nucleotide −1 at the cleavage position. Biochemistry 40, 3363–3369 (2001).

    CAS  PubMed  Google Scholar 

  47. Christian, E.L. & Harris, M.E. The track of the pre-tRNA 5′ leader in the ribonuclease P ribozyme-substrate complex. Biochemistry 38, 12629–12638 (1999).

    CAS  PubMed  Google Scholar 

  48. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  PubMed  Google Scholar 

  49. Brown, J.W. The ribonuclease P database. Nucleic Acids Res. 27, 314 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank A. Krivenko for cloning and initial characterization of the catalytic-domain constructs. We thank R. Batey for helpful comments regarding this work. The research was supported by The US National Institutes of Health (grant GM-34527 to N.R.P. and Molecular Biophysics Training Grant T32 GM-65103 to A.H.B.) through the University of Colorado at Boulder.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado, Boulder, 80309, Colorado, USA

    Amy H Buck

  2. Department of Molecular Cellular and Developmental Biology, University of Colorado, Boulder, 80309, Colorado, USA

    Alexei V Kazantsev, Andrew B Dalby & Norman R Pace

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Correspondence to Norman R Pace.

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Supplementary information

Supplementary Table 1

Phosphorothioate-I2 footprint of RNase P RNAs in the presence of RNase P proteins. (PDF 25 kb)

Supplementary Table 2

Difference in phosphorothioate-I2 cleavage of residues in the I and N folding states of RNase P RNA. (PDF 40 kb)

Supplementary Video 1

Full rotational view of the ternary complex model. (MOV 1780 kb)

Supplementary Methods (PDF 66 kb)

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Buck, A., Kazantsev, A., Dalby, A. et al. Structural perspective on the activation of RNase P RNA by protein. Nat Struct Mol Biol 12, 958–964 (2005). https://doi.org/10.1038/nsmb1004

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