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

Early transcriptional regulation is a major kinetic bottleneck in gene expression, yet the dynamics of RNA polymerase II in living cells remain poorly understood. Here, we use single-molecule imaging to quantify RNA polymerase II chromatin engagement in live human cells. We find that RNA polymerase II infrequently enters a long-lived binding state associated with productive elongation, with ~94% of chromatin-associated events dissociating within tens of seconds. Single-cell analysis reveals substantial heterogeneity in the fraction of RNA polymerase II reaching productive elongation. Spatial heatmap analysis identifies RNA polymerase II clusters enriched for repeated long-lived binding events whose abundance is selectively perturbed by transcriptional inhibitors. Acute degradation of the TFIID subunit TAF1 reduces RNA polymerase II clustering and increases the global pausing index, supporting a model in which TAF1 promotes pause release and entry into productive elongation. Together, these findings provide a single-molecule framework for quantifying transcription kinetics in individual living cells.