In vivo dynamics of RNA polymerase II transcription.
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.
Darzacq
Xavier
X
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Shav-Tal
Yaron
Y
de Turris
Valeria
V
Brody
Yehuda
Y
Shenoy
Shailesh M
SM
Phair
Robert D
RD
Singer
Robert H
RH
D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007700:Kinetics; D010766:Phosphorylation; D010777:Photochemistry; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D017931:DNA Primers; D012319:RNA Polymerase II