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J. Biol. Chem., Vol. 282, Issue 15, 11047-11057, April 13, 2007

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The RPB7 Orthologue E' Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation^*

From the Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universität Regensburg, 93053 Regensburg, Germany

RNA polymerases from Archaea and Eukaryotes consist of a core enzyme associated with a dimeric E'F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the core enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The core enzyme showed significant activity between 70 and 80 °C but was almost inactive at 60 °C. E' stimulated the activity of the core enzyme at 60 °C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the core enzyme, subunit E', and of archaeal TFE to open complex formation. Opening in the –2 and –4 region could be achieved by the core enzyme, subunit E' stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E'-independent role of TFE in the stabilization of open complexes.

Received for publication, December 20, 2006 , and in revised form, February 16, 2007.

^* This work was supported by the priority program "Genome Function and Regulation" of the Deutsche Forschungsgemeinschaft. 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.

¹ To whom correspondence should be addressed: Universitaetsstrasse 31, D-93053 Regensburg, Germany. Tel.: 49-941-943-3160; Fax: 49-941-943-2403; E-mail: michael.thomm{at}biologie.uni-regensburg.de.

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