Transcription initiation by human RNA polymerase II visualized at single-molecule resolution.
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.
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
Andrey
A
Revyakin
Zhengjian
Z
Zhang
Robert A
RA
Coleman
Yan
Y
Li
Carla
C
Inouye
Julian K
JK
Lucas
Sang-Ryul
SR
Park
Steven
S
Chu
Robert
R
Tjian
D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D009154:Mutation; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000378:metabolism; D035362:Transcription Factor TFIID / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D016329:Sp1 Transcription Factor; D035362:Transcription Factor TFIID; D012319:RNA Polymerase II