Summary Cells are built from vast networks of competing molecular interactions, most of which have been impossible to monitor in vivo. We recently devised a new strategy, proximity-assisted photoactivation (PAPA), to detect these interactions at single-molecule resolution in live cells. Here we apply PAPA to visualize the network of interactions that regulate the central transcription elongation factor P-TEFb. PAPA between multiple pairs of endogenous proteins, combined with fast single-molecule tracking (fSMT), revealed that inactive P-TEFb within the 7SK ribonucleoprotein complex is largely unbound to chromatin, that this complex dissociates within minutes of treatment with a P-TEFb kinase inhibitor, and that heterogeneous ribonucleoproteins (hnRNPs) bind 7SK concomitant with P-TEFb release. Unlike 7SK-bound P-TEFb, P-TEFb associated with the coactivator BRD4 exhibited increased binding to chromatin. Our results address longstanding questions about a key transcriptional regulator and demonstrate that PAPA-fSMT can probe subunit interactions and exchange within endogenous regulatory complexes in live cells.

Single-molecule live imaging of subunit interactions and exchange within cellular regulatory complexes