{"doi":"10.1101/2023.02.28.530422","title":"Cryo-EM-guided engineering of T-box-tRNA modules with enhanced selectivity and sensitivity in translational regulation","abstract":"Abstract Riboswitches are non-coding RNA elements that play vital roles in regulating gene expression. Their specific ligand-dependent structural reorganization facilitates their use as templates for design of engineered RNA switches for therapeutics, nanotechnology and synthetic biology. T-box riboswitches bind tRNAs to sense aminoacylation and control gene expression via transcription attenuation or translation inhibition. Here we determine the cryo-EM structure of the wild-type Mycobacterium smegmatis ileS T-box in complex with its cognate tRNA Ile . This structure shows a very flexible antisequestrator region that tolerates both 3’-OH and 2’,3’-cyclic phosphate modification at the 3’ end of tRNA Ile . Elongation of one helical turn (11-base pair) in both the tRNA acceptor arm and T-box Stem III maintains T-box-tRNA complex formation and increases the selectivity for tRNA 3’ end modification. Moreover, elongation of Stem III results in ∼6-fold tighter binding to tRNA, which leads to increased sensitivity of downstream translational regulation indicated by precedent translation. Our results demonstrate that cryo-EM can guide RNA engineering to design improved riboswitch modules for translational regulation, and potentially a variety of additional functions.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":392222,"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":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9509,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":737024,"name":"Chong Zhang","orcid":"0000-0001-9609-8855","position":1,"is_corresponding":false},{"id":634744,"name":"Bingnan Luo","orcid":"0000-0002-9367-4359","position":2,"is_corresponding":false},{"id":1165831,"name":"Jane K. Frandsen","orcid":null,"position":3,"is_corresponding":false},{"id":58890,"name":"Andrew M. Watkins","orcid":"0000-0003-1617-1720","position":4,"is_corresponding":false},{"id":1165832,"name":"Kaiyu Li","orcid":null,"position":5,"is_corresponding":false},{"id":1165461,"name":"Minghua Zhang","orcid":"0000-0002-1927-5405","position":6,"is_corresponding":false},{"id":1165462,"name":"Xiawei Wei","orcid":"0000-0002-4521-6150","position":7,"is_corresponding":false},{"id":1165463,"name":"Yang Yang","orcid":"0009-0009-3902-7570","position":8,"is_corresponding":false},{"id":1165464,"name":"Tina M. Henkin","orcid":"0000-0002-0063-7488","position":9,"is_corresponding":false},{"id":228229,"name":"Zhaoming Su","orcid":"0000-0002-9279-1721","position":10,"is_corresponding":false},{"id":1165460,"name":"Xinyu Jia","orcid":"0000-0002-0962-2258","position":0,"is_corresponding":true}],"reference_count":44,"raw_metadata":null,"created_at":"2026-07-19T01:18:52.232318Z","pmid":"36909519","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":[]}