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bioRxiv · 10.1101/2024.06.25.600644

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

Graham, T. G. W.·Dugast-Darzacq, C.·Dailey, G. M.·Weng, B.·Darzacq, X.·Tjian, R.
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Abstract

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.

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Graham, T. G. W., Dugast-Darzacq, C., Dailey, G. M., Weng, B., Darzacq, X., Tjian, R.. 2024-06-25. Single-molecule live imaging of subunit interactions and exchange within cellular regulatory complexes. https://doi.org/10.1101/2024.06.25.600644

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