In vivo dynamics of RNA polymerase II transcription

Xavier Darzacq, Yaron Shav-Tal, Valeria De Turris, Yehuda Brody, Shailesh M. Shenoy, Robert D. Phair, Robert H. Singer

Research output: Contribution to journalArticle

388 Citations (Scopus)

Abstract

We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min-1, much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.

Original languageEnglish (US)
Pages (from-to)796-806
Number of pages11
JournalNature Structural and Molecular Biology
Volume14
Issue number9
DOIs
StatePublished - Sep 2007

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RNA Polymerase II
Photobleaching
Messenger RNA
Systems Analysis
Genes
DNA
Enzymes
Population
Proteins

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

In vivo dynamics of RNA polymerase II transcription. / Darzacq, Xavier; Shav-Tal, Yaron; De Turris, Valeria; Brody, Yehuda; Shenoy, Shailesh M.; Phair, Robert D.; Singer, Robert H.

In: Nature Structural and Molecular Biology, Vol. 14, No. 9, 09.2007, p. 796-806.

Research output: Contribution to journalArticle

Darzacq, X, Shav-Tal, Y, De Turris, V, Brody, Y, Shenoy, SM, Phair, RD & Singer, RH 2007, 'In vivo dynamics of RNA polymerase II transcription', Nature Structural and Molecular Biology, vol. 14, no. 9, pp. 796-806. https://doi.org/10.1038/nsmb1280
Darzacq X, Shav-Tal Y, De Turris V, Brody Y, Shenoy SM, Phair RD et al. In vivo dynamics of RNA polymerase II transcription. Nature Structural and Molecular Biology. 2007 Sep;14(9):796-806. https://doi.org/10.1038/nsmb1280
Darzacq, Xavier ; Shav-Tal, Yaron ; De Turris, Valeria ; Brody, Yehuda ; Shenoy, Shailesh M. ; Phair, Robert D. ; Singer, Robert H. / In vivo dynamics of RNA polymerase II transcription. In: Nature Structural and Molecular Biology. 2007 ; Vol. 14, No. 9. pp. 796-806.
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