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 pro tein-only R Nase 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 μM 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.

Mode of inhibition of RNase P by gambogic acid and juglone