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
Part of the Methods in Molecular Biology book series (MIMB, volume 1520)


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 



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.


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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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