{"doi":"10.1101/2022.10.20.512141","title":"Mode of inhibition of RNase P by gambogic acid and juglone","abstract":"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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":312608,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9584,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":419253,"name":"Agnes Karasik","orcid":null,"position":1,"is_corresponding":false},{"id":476587,"name":"Kipchumba Kaitany","orcid":"0000-0003-4085-5399","position":2,"is_corresponding":false},{"id":63441,"name":"Carol A. Fierke","orcid":"0000-0002-1481-0579","position":3,"is_corresponding":false},{"id":418435,"name":"Markos Koutmos","orcid":"0000-0003-0933-6312","position":4,"is_corresponding":false},{"id":943687,"name":"Nancy Wu Meyers","orcid":null,"position":0,"is_corresponding":true}],"reference_count":49,"raw_metadata":null,"created_at":"2026-07-19T00:33:36.072096Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}