Real-time observation of the initiation of RNA polymerase II transcription.
Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.
Department of Applied Physics, Stanford University, Stanford, California 94305, USA.
Furqan M
FM
Fazal
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Cong A
CA
Meng
Department of Structural Biology, Stanford University, Stanford, California 94305, USA.
Kenji
K
Murakami
Department of Structural Biology, Stanford University, Stanford, California 94305, USA.
Roger D
RD
Kornberg
Department of Applied Physics, Stanford University, Stanford, California 94305, USA.|Department of Biology, Stanford University, Stanford, California 94305, USA.
Steven M
SM
Block
D020029:Base Pairing / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D052898:Optical Tweezers; D011401:Promoter Regions, Genetic / Q000235:genetics; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D051758:Transcription Factor TFIIH / Q000378:metabolism; D061785:Transcription Initiation, Genetic
D012333:RNA, Messenger; D051758:Transcription Factor TFIIH; D004247:DNA; D012319:RNA Polymerase II