Chapter title |
Fluorescence-Based Real-Time Activity Assays to Identify RNase P Inhibitors
|
---|---|
Chapter number | 12 |
Book title |
Antibiotics
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6634-9_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6632-5, 978-1-4939-6634-9
|
Authors |
Yu Chen, Xin Liu, Nancy Wu, Carol A. Fierke, Chen, Yu, Liu, Xin, Wu, Nancy, Fierke, Carol A. |
Editors |
Peter Sass |
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-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. |
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