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| The EMBO Journal
(1997) 16, 7468–7480, doi:10.1093/emboj/16.24.7468 |
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| Three transitions in the RNA polymerase II transcription complex during initiation |
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| Frank C. P. Holstege2, Ulrike Fiedler1 and H. Th. Marc Timmers1 |
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1 Laboratory for Physiological Chemistry, Utrecht University, PO Box 80042, 3508 TA Utrecht, The Netherlands 2 Present address: Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142, USA To whom correspondence should be addressed H. Th. Marc Timmers, H.T.M.TIMMERS@med.ruu.nl Received 18 July 1997; Revised 6 October 1997. |
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| Abstract |
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We have analyzed transcription initiation by RNA polymerase II (pol II) in a highly efficient in vitro transcription system composed of essentially homogeneous protein preparations. The pol II complex was stalled on adenovirus major late promoter templates at defined positions, and the open region and RNA products of these complexes were examined. The first transition is formation of the open complex, which can be reversed by addition of ATP S. The open region is no longer sensitive to ATPS after formation of a four-nucleotide RNA, which constitutes the second transition. This indicates that the ATP-dependent DNA helicase activity of TFIIH is required to maintain the open region only during formation of the first three phosphodiester bonds. The downstream part of the transcription bubble expands in a continuous motion, but the initially opened region (-9/-2 on the non-template strand) recloses abruptly when transcription reaches register 11. This third transition is accompanied by a switch from abortive to productive RNA synthesis, which implies promoter clearance. Our findings provide a framework to analyze regulation of these specific transitions during transcription initiation by pol II. |
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| Keywords: abortive transcription, DNA helicase, RNA polymerase II, TFIIH, transcription initiation |
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