title: Fluorescence-Based Real-Time Activity Assays to Identify RNase P Inhibitors.
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-tRNAAspsubstrate. 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.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Chemical Biology Program, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: Wu
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA. fierke@umich.edu.|Chemical Biology Program, University of Michigan, Ann Arbor, MI, 48109, USA. fierke@umich.edu.|Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA. fierke@umich.edu.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D007700:Kinetics; D012322:RNA Precursors / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D043262:Ribonuclease P / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D012322:RNA Precursors; D012329:RNA, Bacterial; D012343:RNA, Transfer; D043262:Ribonuclease P
keywords: Antibiotics; Fluorescence anisotropy; Fluorescence polarization; High-throughput screening; Inhibitor; Mode of inhibition; Neomycin; RNase P; tRNA