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Nucleic Acids Research Advance Access originally published online on July 20, 2009
Nucleic Acids Research 2009 37(17):5803-5809; doi:10.1093/nar/gkp601
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Nucleic Acids Research, 2009, Vol. 37, No. 17 5803-5809
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex

Joanna Andrecka1, Barbara Treutlein1, Maria Angeles Izquierdo Arcusa2, Adam Muschielok1, Robert Lewis1, Alan C. M. Cheung1,3, Patrick Cramer1,3 and Jens Michaelis1,*

1Department of Chemistry and Biochemistry and Center for Integrated Protein Science München, Ludwig-Maximilians-Universität München, Butenandtstr.11, 81377 München, Germany, 2Departamento de Quimica Inorganica y Organica, Universidad Jaume I de Castellon, 12071 Castellon, Spain and 3Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor Lynen Str. 25, 81377 München, Germany

*To whom correspondence should be addressed. Tel: +49 89 2180 77561; Fax: +49 89 2180 77560; Email: michaelis{at}lmu.de

Received May 15, 2009. Revised June 30, 2009. Accepted July 1, 2009.

Crystallographic studies of the RNA polymerase II (Pol II) elongation complex (EC) revealed the locations of downstream DNA and the DNA-RNA hybrid, but not the course of the nontemplate DNA strand in the transcription bubble and the upstream DNA duplex. Here we used single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The nontemplate strand passes loop β10-β11 on the Pol II lobe, and then turns to the other side of the cleft above the rudder. The upstream DNA duplex exits at an approximately right angle from the incoming downstream DNA, and emanates from the cleft between the protrusion and clamp. Comparison with published data suggests that the architecture of the complete EC is conserved from bacteria to eukaryotes and that upstream DNA is relocated during the initiation–elongation transition.


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