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J. Biol. Chem., Vol. 282, Issue 30, 21578-21582, July 27, 2007

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Multisubunit RNA Polymerases Melt Only a Single DNA Base Pair Downstream of the Active Site^*

Ekaterina Kashkina, Michael Anikin, Florian Brueckner^¶, Elisabeth Lehmann^¶, Sergey N. Kochetkov, William T. McAllister, Patrick Cramer^¶, and Dmitry Temiakov¹

From the Department of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084, and the Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991, Moscow, Russian Federation, and the ^¶Gene Center Munich and Center for integrated Protein Science, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universitaät München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany

To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site.

Received for publication, May 17, 2007 , and in revised form, May 24, 2007.

^* This work was supported by a University of Medicine and Dentistry of New Jersey Foundation Grant (to D. T.), by National Institutes of Health Grant GM38147 (to W. T. M), and by grants from the Deutsche Forschungsgemeinschaft, the SFB646, the Center for integrated Protein Science, and the Fonds der Chemischen Industrie (to P. C.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains four supplemental figures.

¹ To whom correspondence should be addressed: E-mail: d.temiakov{at}umdnj.edu.

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