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bioRxiv · 10.1101/2022.10.20.512141

Mode of inhibition of RNase P by gambogic acid and juglone

Wu, N.·Karasik, A.·Kaitany, K.·Koutmos, M.·Fierke, C. A.
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Abstract

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5 end of precursor transfer RNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein (RNP) complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of protein-only RNase P that can be used as tools for studying the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization (FP) cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 M range were identified and analyzed. These compounds similarly inhibit human mtRNase P, a multi-subunit protein enzyme, and are 50-fold less potent against bacterial RNA-dependent RNase P. Biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor that targets the PRORP1-substrate complex while juglone acts as time-dependent inhibitor of PRORP1. X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. A model consistent with the kinetic data is that juglone binds to PRORP1 rapidly to form an inactive enzyme-inhibitor (EI) complex, and then undergoes a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

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Wu, N., Karasik, A., Kaitany, K., Koutmos, M., Fierke, C. A.. 2022-10-21. Mode of inhibition of RNase P by gambogic acid and juglone. https://doi.org/10.1101/2022.10.20.512141

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