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

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-tRNA^Asp 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.