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.
Description
In vivo dynamics of RNA polymerase II transcriptio... [Nat Struct Mol Biol. 2007] - PubMed result
%0 Journal Article
%1 darzacq2007dynamics
%A Darzacq, X
%A Shav-Tal, Y
%A de Turris, V
%A Brody, Y
%A Shenoy, S M
%A Phair, R D
%A Singer, R H
%D 2007
%J Nat Struct Mol Biol
%K polymerase stochastic_transcription
%N 9
%P 796-806
%R 10.1038/nsmb1280
%T In vivo dynamics of RNA polymerase II transcription
%U http://www.ncbi.nlm.nih.gov/pubmed/17676063
%V 14
%X 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.
@article{darzacq2007dynamics,
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.},
added-at = {2010-05-06T20:55:39.000+0200},
author = {Darzacq, X and Shav-Tal, Y and de Turris, V and Brody, Y and Shenoy, S M and Phair, R D and Singer, R H},
biburl = {https://www.bibsonomy.org/bibtex/2452318a34040d337dcf5340cd0e2c0c7/peter.ralph},
description = {In vivo dynamics of RNA polymerase II transcriptio... [Nat Struct Mol Biol. 2007] - PubMed result},
doi = {10.1038/nsmb1280},
interhash = {d2fd73e72f500bb27b136a2e47937be2},
intrahash = {452318a34040d337dcf5340cd0e2c0c7},
journal = {Nat Struct Mol Biol},
keywords = {polymerase stochastic_transcription},
month = sep,
number = 9,
pages = {796-806},
pmid = {17676063},
timestamp = {2010-05-06T20:55:39.000+0200},
title = {{{\it In vivo}} dynamics of {RNA} polymerase {II} transcription},
url = {http://www.ncbi.nlm.nih.gov/pubmed/17676063},
volume = 14,
year = 2007
}