{"doi":"10.1261/rna.080120.124","title":"Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2′-phosphate end","abstract":"Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3′-OH,2′-PO 4 and 5′-PO 4 RNA ends to form a 2′-PO 4 ,3′-5′ phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5′-PO 4 RNA terminus to form an RNA-adenylate intermediate (A 5′ pp 5′ RNA). Third, LIG directs the attack of an RNA 3′-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2′-PO 4 to synthesize a 3′–5′ phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2′-PO 4 . Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA 2′ p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His–Arg–Arg triad as the enforcer of the 2′-PO 4 end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.","journal":"RNA","year":2024,"id":483544,"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":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9571,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":409034,"name":"Stewart Shuman","orcid":"0000-0001-5034-6438","position":1,"is_corresponding":false},{"id":411733,"name":"Shreya Ghosh","orcid":"0000-0002-1709-8011","position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":null,"created_at":"2026-07-19T02:07:33.718973Z","pmid":"39013577","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":[]}