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Antibiotics pp 201-225 | Cite as

Fluorescence-Based Real-Time Activity Assays to Identify RNase P Inhibitors

  • Yu Chen
  • Xin Liu
  • Nancy Wu
  • Carol A. Fierke
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1520)

Abstract

Transfer RNA is transcribed as precursor molecules that are processed before participating in translation catalyzed by the ribosome. Ribonuclease P is the endonuclease that catalyzes the 5′ end maturation of precursor tRNA and it is essential for cell survival. Bacterial RNase P has a distinct subunit composition compared to the eukaryal counterparts; therefore, it is an attractive antibacterial target. Here, we describe a real-time fluorescence-based RNase P activity assay using fluorescence polarization/anisotropy with a 5′ end fluorescein-labeled pre-tRNAAsp substrate. This FP/FA assay is sensitive, robust, and easy to transition to a high-throughput mode and it also detects ligands that interact with pre-tRNA. We apply this FP/FA assay to measure Bacillus subtilis RNase P activity under single and multiple turnover conditions in a continuous format and a high-throughput screen of inhibitors, as well as determining the dissociation constant of pre-tRNA for small molecules.

Key words

RNase P Fluorescence polarization Fluorescence anisotropy tRNA High-throughput screening Inhibitor Antibiotics Neomycin Mode of inhibition 

Notes

Acknowledgment

This work was supported by grants from National Institute of Health [R01 GM55387 to C.A.F.], Pilot Screen Grant from the Center for Chemical Genomics at the University of Michigan [to C.A.F.], Rackham Graduate Student Research Grant [to X.L.]. Thanks go to Drs. Elaina Zverina, Lyra Chang, John Hsieh and Daina Zeng for helpful discussions on the development of the HTS assay and Professors Jason Gestwicki and Anna Mapp for sharing plate-reader instruments. We thank Martha Larsen, Steven Swaney, and Paul Kirchhoff at the Center for Chemical Genomics (CCG) at University of Michigan for their help with the compound library screen and data mining.

References

  1. 1.
    Hartmann RK, Gossringer M, Spath B, Fischer S, Marchfelder A (2009) The making of tRNAs and more—RNase P and tRNase Z. Prog Nucleic Acid Res Mol Biol 85:319–368Google Scholar
  2. 2.
    Iwata-Reuyl D (2008) An embarrassment of riches: the enzymology of RNA modification. Curr Opin Chem Biol 12(2):126–133CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Xiong Y, Steitz TA (2006) A story with a good ending: tRNA 3′-end maturation by CCA-adding enzymes. Curr Opin Struct Biol 16(1):12–17CrossRefPubMedGoogle Scholar
  4. 4.
    Phizicky EM, Hopper AK (2010) tRNA biology charges to the front. Genes Dev 24(17):1832–1860CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kazantsev AV, Pace NR (2006) Bacterial RNase P: a new view of an ancient enzyme. Nat Rev Microbiol 4(10):729–740CrossRefPubMedGoogle Scholar
  6. 6.
    Smith JK, Hsieh J, Fierke CA (2007) Importance of RNA-protein interactions in bacterial ribonuclease P structure and catalysis. Biopolymers 87(5-6):329–338CrossRefPubMedGoogle Scholar
  7. 7.
    Kirsebom LA (2007) RNase P RNA mediated cleavage: substrate recognition and catalysis. Biochimie 89(10):1183–1194CrossRefPubMedGoogle Scholar
  8. 8.
    Harris ME, Christian EL (2003) Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P. Curr Opin Struct Biol 13(3):325–333CrossRefPubMedGoogle Scholar
  9. 9.
    Randau L, Schroder I, Soll D (2008) Life without RNase P. Nature 453(7191):120–123CrossRefPubMedGoogle Scholar
  10. 10.
    Walker SC, Engelke DR (2006) Ribonuclease P: the evolution of an ancient RNA enzyme. Crit Rev Biochem Mol Biol 41(2):77–102CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Apirion D (1980) Genetic mapping and some characterization of the rnpA49 mutation of Escherichia coli that affects the RNA-processing enzyme ribonuclease P. Genetics 94(2):291–299PubMedPubMedCentralGoogle Scholar
  12. 12.
    Eder PS, Hatfield C, Vioque A, Gopalan V (2003) Bacterial RNase P as a potential target for novel anti-infectives. Curr Opin Investig Drugs 4(8):937–943PubMedGoogle Scholar
  13. 13.
    Frank DN, Pace NR (1998) Ribonuclease P: unity and diversity in a tRNA processing ribozyme. Annu Rev Biochem 67:153–180CrossRefPubMedGoogle Scholar
  14. 14.
    Hartmann E, Hartmann RK (2003) The enigma of ribonuclease P evolution. Trends Genet 19(10):561–569CrossRefPubMedGoogle Scholar
  15. 15.
    Hall TA, Brown JW (2001) The ribonuclease P family. Methods Enzymol 341:56–77CrossRefPubMedGoogle Scholar
  16. 16.
    Hsieh J, Andrews AJ, Fierke CA (2004) Roles of protein subunits in RNA-protein complexes: lessons from ribonuclease P. Biopolymers 73(1):79–89CrossRefPubMedGoogle Scholar
  17. 17.
    Lai LB, Vioque A, Kirsebom LA, Gopalan V (2010) Unexpected diversity of RNase P, an ancient tRNA processing enzyme: challenges and prospects. FEBS Lett 584(2):287–296CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Esakova O, Krasilnikov AS (2010) Of proteins and RNA: the RNase P/MRP family. RNA 16(9):1725–1747CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Thomas BC, Gao L, Stomp D, Li X, Gegenheimer PA (1995) Spinach chloroplast RNase P: a putative protein enzyme. Nucleic Acids Symp Ser 33:95–98Google Scholar
  20. 20.
    Gobert A, Butmann B, Taschner A, Gößringer M, Holzmann J, Hartmann RK, Rossmanith W, Giegé P (2010) A single Arabidopsis organellar protein has RNase P activity. Nat Struct Mol Biol 17(6):740–744CrossRefPubMedGoogle Scholar
  21. 21.
    Holzmann J, Frank P, Loffler E, Bennett KL, Gerner C, Rossmanith W (2008) RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. Cell 135(3):462–474CrossRefPubMedGoogle Scholar
  22. 22.
    Trang P, Kim K, Liu F (2004) Developing RNase P ribozymes for gene-targeting and antiviral therapy. Cell Microbiol 6(6):499–508CrossRefPubMedGoogle Scholar
  23. 23.
    Willkomm DK, Gruegelsiepe H, Goudinakis O, Kretschmer-Kazemi Far R, Bald R, Erdmann VA, Hartmann RK (2003) Evaluation of bacterial RNase P RNA as a drug target. Chembiochem 4(10):1041–1048CrossRefPubMedGoogle Scholar
  24. 24.
    Gossringer M, Kretschmer-Kazemi Far R, Hartmann RK (2006) Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression. J Bacteriol 188(19):6816–6823CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Waugh DS, Pace NR (1990) Complementation of an RNase P RNA (rnpB) gene deletion in Escherichia coli by homologous genes from distantly related eubacteria. J Bacteriol 172(11):6316–6322CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kobayashi K, Ehrlich SD, Albertini A, Amati G, Andersen KK, Arnaud M, Asai K, Ashikaga S, Aymerich S, Bessieres P, Boland F, Brignell SC, Bron S, Bunai K, Chapuis J, Christiansen LC, Danchin A, Debarbouille M, Dervyn E, Deuerling E, Devine K, Devine SK, Dreesen O, Errington J, Fillinger S, Foster SJ, Fujita Y, Galizzi A, Gardan R, Eschevins C, Fukushima T, Haga K, Harwood CR, Hecker M, Hosoya D, Hullo MF, Kakeshita H, Karamata D, Kasahara Y, Kawamura F, Koga K, Koski P, Kuwana R, Imamura D, Ishimaru M, Ishikawa S, Ishio I, Le Coq D, Masson A, Mauel C, Meima R, Mellado RP, Moir A, Moriya S, Nagakawa E, Nanamiya H, Nakai S, Nygaard P, Ogura M, Ohanan T, O’Reilly M, O’Rourke M, Pragai Z, Pooley HM, Rapoport G, Rawlins JP, Rivas LA, Rivolta C, Sadaie A, Sadaie Y, Sarvas M, Sato T, Saxild HH, Scanlan E, Schumann W, Seegers JF, Sekiguchi J, Sekowska A, Seror SJ, Simon M, Stragier P, Studer R, Takamatsu H, Tanaka T, Takeuchi M, Thomaides HB, Vagner V, van Dijl JM, Watabe K, Wipat A, Yamamoto H, Yamamoto M, Yamamoto Y, Yamane K, Yata K, Yoshida K, Yoshikawa H, Zuber U, Ogasawara N (2003) Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100(8):4678–4683CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Mikkelsen NE, Brannvall M, Virtanen A, Kirsebom LA (1999) Inhibition of RNase P RNA cleavage by aminoglycosides. Proc Natl Acad Sci U S A 96(11):6155–6160CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Rueda D, Hsieh J, Day-Storms JJ, Fierke CA, Walter NG (2005) The 5′ leader of precursor tRNA(Asp) bound to the Bacillus subtilis RNase P holoenzyme has an extended conformation. Biochemistry 44(49):16130–16139CrossRefPubMedGoogle Scholar
  29. 29.
    Hsieh J, Fierke CA (2009) Conformational change in the Bacillus subtilis RNase P holoenzyme—pre-tRNA complex enhances substrate affinity and limits cleavage rate. RNA 15(8):1565–1577CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Hsieh J, Koutmou KS, Rueda D, Koutmos M, Walter NG, Fierke CA (2010) A divalent cation stabilizes the active conformation of the B. subtilis RNase P x pre-tRNA complex: a role for an inner-sphere metal ion in RNase P. J Mol Biol 400(1):38–51CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Giordano T, Sturgess MA, Rao SJ (2006) Inhibitors of RNase P proteins as antibacterial compounds. Google PatentsGoogle Scholar
  32. 32.
    Liu X, Chen Y, Fierke CA (2014) A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P. Nucleic Acids Res 42(20), e159CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Jameson DM, Ross JA (2010) Fluorescence polarization/anisotropy in diagnostics and imaging. Chem Rev 110(5):2685–2708CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Behrman EJ (2000) An improved synthesis of guanosine 5′-monothiophosphate. J Chem Res 2000(9):446–447CrossRefGoogle Scholar
  35. 35.
    Beebe JA, Fierke CA (1994) A kinetic mechanism for cleavage of precursor tRNA(Asp) catalyzed by the RNA component of Bacillus subtilis ribonuclease P. Biochemistry 33(34):10294–10304CrossRefPubMedGoogle Scholar
  36. 36.
    He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK (1997) Rapid mutagenesis and purification of phage RNA polymerases. Protein Expr Purif 9(1):142–151CrossRefPubMedGoogle Scholar
  37. 37.
    Niranjanakumari S, Kurz JC, Fierke CA (1998) Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis. Nucleic Acids Res 26(13):3090–3096CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Niranjanakumari S, Stams T, Crary SM, Christianson DW, Fierke CA (1998) Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA. Proc Natl Acad Sci U S A 95(26):15212–15217CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Mocz G, Helms MK, Jameson DM, Gibbons IR (1998) Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2′(3′)-O-(-N-Methylanthraniloyl)-adenosine 5′-triphosphate. Biochemistry 37(27):9862–9869CrossRefPubMedGoogle Scholar
  40. 40.
    Sem DS, McNeeley PA (1999) Application of fluorescence polarization to the steady-state enzyme kinetic analysis of calpain II. FEBS Lett 443(1):17–19CrossRefPubMedGoogle Scholar
  41. 41.
    Michaelis L, Menten ML, Johnson KA, Goody RS (2011) The original Michaelis constant: translation of the 1913 Michaelis-Menten paper. Biochemistry 50(39):8264–8269CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73CrossRefPubMedGoogle Scholar
  43. 43.
    Seidler J, McGovern SL, Doman TN, Shoichet BK (2003) Identification and prediction of promiscuous aggregating inhibitors among known drugs. J Med Chem 46(21):4477–4486CrossRefPubMedGoogle Scholar
  44. 44.
    Copeland RA (2000). Enzymes: a practical introduction to structure, mechanism, and data analysis. Wiley-VCH Inc.: 266–349Google Scholar
  45. 45.
    Prinz H (2010) Hill coefficients, dose-response curves and allosteric mechanisms. J Chem Biol 3(1):37–44CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Yu Chen
    • 1
  • Xin Liu
    • 1
  • Nancy Wu
    • 2
  • Carol A. Fierke
    • 1
    • 2
    • 3
  1. 1.Department of ChemistryUniversity of MichiganAnn ArborUSA
  2. 2.Chemical Biology ProgramUniversity of MichiganAnn ArborUSA
  3. 3.Department of Biological ChemistryUniversity of MichiganAnn ArborUSA

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