Graham Thomas G W, Dugast-Darzacq Claire, Dailey Gina M, Weng Britney, Anantakrishnan Sathvik, Darzacq Xavier, Tjian Robert
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Mol Cell. 2025 Aug 7;85(15):2854-2868.e7. doi: 10.1016/j.molcel.2025.06.028. Epub 2025 Jul 23.
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.
细胞由庞大的相互依赖的分子相互作用网络构成。在此，我们将邻近辅助光激活（PAPA）与自动快速单分子追踪（fSMT）相结合，以探测活的人类细胞内源性蛋白质复合物中的亚基相互作用。PAPA - fSMT显示，无活性的正性转录延伸因子b（P - TEFb）：7SK核糖核蛋白复合物主要是可移动的，未与染色质相连，并检测到特定的异质性核核糖核蛋白（hnRNPs）与7SK复合物的相互作用。细胞周期蛋白依赖性激酶9（Cdk9）的抑制作用使hnRNP R从大RNA中释放出来，增加了hnRNP R与7SK的结合，并在数分钟内将P - TEFb从7SK中逐出，这与通过竞争性蛋白质 - RNA相互作用对P - TEFb进行快速、稳态的负反馈调节相一致。与共激活因子BRD4的结合增加了P - TEFb与染色质的结合，这依赖于BRD4的溴结构域。最后，PAPA检测到HIV转录激活因子Tat使P - TEFb从7SK中释放。我们的结果揭示了P - TEFb调控中以前在活细胞中无法获得的方面，并开辟了一条探测内源性调节复合物中亚基相互作用和交换的途径。
