Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
Genes & development (Impact Factor: 10.8). 07/2012; 26(15):1691-702. DOI: 10.1101/gad.194936.112
Source: PubMed


Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

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    • "These methods offer the promise of tracking the movements and behaviors of individual transcription factors as they search for cognate binding sites on interphase chromatin within the nucleus of individual living cells in subsecond real-time measurements (Abrahamsson et al., 2013; Betzig et al., 2006; Gao et al., 2012; Huang et al., 2009; Huisken et al., 2004; Shao et al., 2011; Wu et al., 2013). When combined with genetic manipulations, genome-wide analysis, and in vitro single-molecule assays (Revyakin et al., 2012), these methods can provide extraordinarily quantitative measurements with remarkable temporal and spatial resolution. It is now possible to accurately measure on/off rates, dwell times, 3D diffusion intervals, and search times for individual transcription factors or combinations of transcription factors. "
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    • "In Vitro TIRF Single - Molecule Imaging TIRF microscope - imaging system setup and fluorescent molecule spots ' colocalization analysis were essentially as described previously ( Revyakin et al . , 2012 ) . See the Extended Experimental Procedures for experimental details and imaging analysis ."
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    • "Optical trapping experiments have also recently provided insight into the mechanisms of Pol II elongation (Larson et al. 2012), stalling (Galburt et al. 2007), and interactions with nucleosomes (Hodges et al. 2009). Combining a nanomanipulation method with the single-molecule fluorescencebased detection of component presence as described by Revyakin et al. (2012) could help address questions of timing of complex activity as it relates to complex assembly. The main obstacle here is the required throughput: As eukaryotic transcription initiation is not a deterministic process, one must be able to watch large numbers of DNA molecules in order to detect with statistical significance the 20%–40% fraction that initiates transcription. "
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