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.
Figure 1. Detecting transcription in vivo using fluorescence microscopy
(a) Schematic of the gene cassette17 stably integrated into chromosomes of human U2OS cells. P above protein sequence denotes Pol II phosphorylation state (red, phosphorylated). Reverse tet transactivator (rtTA) in the presence of doxycycline drives gene expression from a minimal CMV promoter17. Arrows indicate the 3.3-kb region transcribed by Pol II and the 2.3-kb region labeled by GFP-MS2 fusion proteins. Red lines indicate targets of FISH oligonucleotide probes. (b–m) Active transcription sites recruit Pol II. In b,e,h,k, RFP-LacI labels gene locus. Immunofluorescence (using indicated antibodies) reveals Pol II in three phosphorylation states: unphosphorylated (c), phosphorylated at Ser5 (f) and phosphorylated at Ser2 (i). l shows that the transcription site recruits YFP–Pol II (YFP-RPB1
Amr). In n–y, nascent mRNAs were detected at active sites. In n,r,v, CFP-LacI labels gene locus. In o,s, mRNAs bound by GFP-MS2 were detected by FISH (probes at 5' and 3' ends are shown in p,t). FISH signals at exon (w) and intron regions (x) colocalize only at transcription site (see merge of each row, q,u,y). Scale bars, 5 
m.
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