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Nat Struct Mol Biol. 2007 Sep;14(9):796-806. Epub 2007 Aug 5.

In vivo dynamics of RNA polymerase II transcription.

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  • 1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

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.

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PMID:
17676063
[PubMed - indexed for MEDLINE]
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