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
1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
2 Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, UMR-8541, Ecole Normale Supérieure, 75005 Paris, France.
3 The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel.
4 Integrative Bioinformatics, Inc., Los Altos, California 94024, USA.
Correspondence should be addressed to Robert H Singer rhsinger@aecom.yu.edu
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.
