In vivo dynamics of RNA polymerase II transcription

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.Transcription by RNA polymerase II (Pol II) is at the core of gene expression and hence is the basis of all cellular activities. Little information exists about the kinetics of this process in live cells 1 , as understanding of gene expression regulation comes from studies using purified proteins. For instance, the subunits of the elongating Pol II are well known 2 and the crystal structure of this enzyme explains much of its behavior in vitro 3,4 . mRNA transcription can be deconstructed into a succession of steps: promoter assembly, clearanceCorrespondence should be addressed to R.H.S. (rhsinger@aecom.yu.edu). AUTHOR CONTRIBUTIONS All data were initially acquired by X.D. and Y.S.-T. Subsequent data were obtained by V.d.T. (Fig. 4a,b and Fig. 5a) and Y.B. (Fig. 9b). S.M.S. was responsible for the microscopy, built the wide-field microscope for live-cell imaging and wrote analysis software. X.D. performed the kinetic modeling. R.D.P. provided consultation on model formulation and testing, and training in the use of the ProcessDB software. R.H.S. supervised the project. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/nsmb/. HHS Public AccessAuthor manuscript Nat Struct Mol Biol. Author manuscript; available in PMC 2016 July 12. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript and escape 5 , followed by elongation and termination. The process of transcriptional initiation involves several structural changes in the polymerase as the nascent transcript elongates 6 . Early in initiation, the polymerase can produce abortive transcripts 7,8 . These abortive cycles have been observed with a single prokaryote polymerase (RNAP) releasing several transcripts without escaping the promoter 9,10 . The elongation step can be regulated by pausing for various times, as demonstrated using prokaryotic polymerases in vitro 11,12 .For eukaryotic cells, attempts have been mad...

Coactivators and general transcription factors have two distinct dynamic populations dependent on transcription

“…First, we chose RPB1 (the largest subunit of Pol II) known to have stable interactions with chromatin (Kimura et al , 2002; Hieda et al , 2005; Boireau et al , 2007; Darzacq et al , 2007; Fromaget & Cook, 2007) and also highly dynamic nuclear factors, like the TFIID subunit TAF5 and the GTF TFIIB (Chen et al , 2002; Sprouse et al , 2008; de Graaf et al , 2010; Ihalainen et al , 2012). In addition, eGFP was used as a control for a freely diffusing protein that should exhibit no specific interactions with the nuclear environment.…”

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

“…A major conclusion of this study is that cleavage/polyadenylation factors are maximally recruited at the 3′ pause 0.5-1.5 kb downstream of poly (A) sites. This downstream pause detected by ChIP may correspond to the pause in live cells detected by FRAP in the 3′ portion of a reporter gene 47 . The 3′ pause was normally followed by termination (Figs.…”

Evidence‐Based Theory for Integrated Genome Regulation of Ontogeny—An Unprecedented Role of Nuclear FGFR1 Signaling

“…During transcriptional activation, the rate of oscillations is further reduced (“S” R1 converts into “ES”, (t/12 > 50 sec), possibly reflecting FGFR1 and RSK1 binding events and the formation of productive elongating complexes. The kinetics of RNA Polymerase II (RPII) are based on the methods of (Darzacq et al, 2007) and are similar to FGFR1. Close co‐localization of nFGFR1 and RNA Pol II is illustrated by immunostaining and super‐resolution microscopy in Video—Supplemental Material (collaborative experiment with Dr. Hari Shroff, NIH) l (Video 1).…”