Single-molecule imaging reveals RNA polymerase II dynamics and TAF1-dependent promoter-proximal pause release

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Transcriptional regulation is a key kinetic bottleneck in gene expression.
• The dynamics of RNA polymerase II (Pol II) in living cells are not well understood.

Purpose of the Study:

• To quantify RNA polymerase II chromatin engagement dynamics in live human cells using single-molecule imaging.
• To investigate the role of TAF1 in regulating transcription kinetics and Pol II behavior.

Main Methods:

• Single-molecule imaging of RNA polymerase II in live human cells.
• Single-cell analysis and spatial heatmap analysis of Pol II binding events.
• Perturbation of TFIID subunit TAF1 via acute degradation.

Main Results:

• RNA polymerase II infrequently enters a long-lived binding state for productive elongation (~94% dissociate within seconds).
• Significant heterogeneity exists in the fraction of Pol II reaching productive elongation across single cells.
• RNA polymerase II clusters, enriched for long-lived binding events, are perturbed by transcriptional inhibitors and reduced by TAF1 degradation.
• TAF1 degradation increases the global pausing index, indicating impaired pause release.

Conclusions:

• A single-molecule framework for quantifying transcription kinetics in individual living cells has been established.
• TAF1 plays a crucial role in promoting RNA polymerase II pause release and entry into productive elongation.
• Findings support a model where TAF1 facilitates efficient transcription by enhancing Pol II clustering and productive elongation.