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Nucleic Acids Research, 2001, Vol. 29, No. 13 2706-2714
© 2001 Oxford University Press

Analysis of the open region of RNA polymerase II transcription complexes in the early phase of elongation

Ulrike Fiedler and H. Th. Marc Timmers* Department of Physiological Chemistry, University Medical Center Utrecht, Stratenum Building, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

The RNA polymerase II (pol II) transcription complex undergoes a structural transition around registers 20–25, as indicated by ExoIII footprinting analyses. We have employed a highly purified system to prepare pol II complexes stalled at very precise positions during the initial stage of transcript elongation. Using potassium permanganate we analyzed the open region (‘transcription bubble’) of complexes stalled between registers 15 and 35. We found that from register 15 up to 25 the transcription bubble expands concomitantly with RNA synthesis. At registers 26 and 27 the bubble has a high tendency to retract at the leading edge. Addition of transcription elongation factor TFIIS re-extends the bubble to the stall site, resulting in complexes competent for transcript elongation. These findings are discussed in the light of the recently determined structures for RNA polymerases.

* To whom correspondence should be addressed. Tel: +31 30 253 8981; Fax: +31 30 253 9035; Email: h.t.m.timmers{at}med.uu.nl Present address:Ulrike Fiedler, Institute for Molecular Oncology, Breisacherstrasse 117, D-79106 Freiburg, Germany


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