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Three transitions in the RNA polymerase II transcription complex during initiation
Author(s) -
Holstege Frank C. P.,
Fiedler Ulrike,
Timmers H. Th. Marc
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.24.7468
Subject(s) - biology , abortive initiation , transcription (linguistics) , rna polymerase ii , transcription factor ii f , transcription factor ii h , transcription bubble , transcription factor ii e , rna polymerase , transcription factor ii b , general transcription factor , polymerase , microbiology and biotechnology , rna , rna polymerase ii holoenzyme , transcription factor ii d , rna helicase a , helicase , dna , genetics , promoter , nucleotide excision repair , gene , gene expression , dna repair , linguistics , philosophy
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 ATPγS. The open region is no longer sensitive to ATPγS 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.