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.
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Xavier
X
Darzacq
Yaron
Y
Shav-Tal
Valeria
V
de Turris
Yehuda
Y
Brody
Shailesh M
SM
Shenoy
Robert D
RD
Phair
Robert H
RH
Singer
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