{"doi":"10.1016/j.molcel.2025.06.028","title":"Single-molecule live imaging of subunit interactions and exchange within cellular regulatory complexes","abstract":"Cells are built from vast networks of interdependent molecular interactions. Here, we combine proximity-assisted photoactivation (PAPA) with automated fast single-molecule tracking (fSMT) to probe subunit interactions within endogenous protein complexes in live human cells. PAPA-fSMT revealed that the inactive positive transcription elongation factor b (P-TEFb):7SK ribonucleoprotein complex is predominantly mobile, not tethered to chromatin, and detected interaction of specific heterogeneous nuclear ribonucleoproteins (hnRNPs) with the 7SK complex. Cyclin-dependent kinase 9 (Cdk9) inhibition liberated hnRNP R from large RNAs, increased hnRNP R binding to 7SK, and evicted P-TEFb from 7SK within minutes-consistent with rapid, homeostatic negative feedback regulation of P-TEFb by competing protein-RNA interactions. Association with the coactivator BRD4 increased P-TEFb chromatin binding, which depended on the BRD4 bromodomains. Finally, PAPA detected the release of P-TEFb from 7SK by the HIV transcriptional activator Tat. Our results illuminate aspects of P-TEFb regulation that were previously inaccessible in live cells and open a route to probe subunit interactions and exchange within endogenous regulatory complexes.","journal":"Molecular Cell","year":2025,"id":517562,"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":8,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9574,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":554004,"name":"Claire Dugast‐Darzacq","orcid":"0000-0001-8602-3534","position":1,"is_corresponding":false},{"id":649431,"name":"Gina M. Dailey","orcid":"0000-0002-8988-963X","position":2,"is_corresponding":false},{"id":1384563,"name":"Britney Weng","orcid":null,"position":3,"is_corresponding":false},{"id":1372110,"name":"Sathvik Anantakrishnan","orcid":"0000-0003-0281-3905","position":4,"is_corresponding":false},{"id":109376,"name":"Xavier Darzacq","orcid":"0000-0003-2537-8395","position":5,"is_corresponding":false},{"id":109375,"name":"Robert Tjian","orcid":"0000-0003-0539-8217","position":6,"is_corresponding":false},{"id":554005,"name":"Thomas G.W. Graham","orcid":"0000-0001-5189-4313","position":0,"is_corresponding":true}],"reference_count":90,"raw_metadata":null,"created_at":"2026-07-19T02:48:59.410472Z","pmid":"40706597","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":[]}