Article abstract
Nature Structural & Molecular Biology 14, 796 - 806 (2007)
Published online: 5 August 2007 | doi:10.1038/nsmb1280
In vivo dynamics of RNA polymerase II transcription
Xavier Darzacq1,2, Yaron Shav-Tal1,3, Valeria de Turris1, Yehuda Brody3, Shailesh M Shenoy1, Robert D Phair4 & Robert H Singer1
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.
- Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
- Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, UMR-8541, Ecole Normale Supérieure, 75005 Paris, France.
- The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel.
- Integrative Bioinformatics, Inc., Los Altos, California 94024, USA.
Correspondence to: Robert H Singer1 e-mail: rhsinger@aecom.yu.edu
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