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Papers In Press, published online ahead of print February 20, 2007
Mikrobiologie, Universität Regensburg, Regensburg 93053
Corresponding Author: michael.thomm{at}biologie.uni-regensburg.de
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 subcomplex 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, -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.
J. Biol. Chem, 10.1074/jbc.M611674200
Submitted on December 20, 2006
Revised on February 16, 2007
Accepted on February 19, 2007
The Rpb7 orthologue E' is required for transcriptional activity of a reconstituted archaeal core enzyme at low temperatures and stimulates open complex formation
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