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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月20日

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  • Nayem Haque,
  • Ronald Cutler,
  • Simone Sidoli,
  • Robert A. Coleman

摘要

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.