Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

Andrey Revyakin, Zhengjian Zhang, Robert A. Coleman, Yan Li, Carla Inouye, Julian K. Lucas, Sang Ryul Park, Steven Chu, Robert Tjian

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

Original languageEnglish (US)
Pages (from-to)1691-1702
Number of pages12
JournalGenes and Development
Volume26
Issue number15
DOIs
Publication statusPublished - Aug 1 2012

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Keywords

  • Pol II transcription
  • Preinitiation complex
  • Reinitiation
  • Single-molecule fluorescence
  • Surface passivation
  • Unstructured probes

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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