The RPB7 orthologue E' is required for transcriptional activity of a reconstituted archaeal core enzyme at low temperatures and stimulates open complex formation.
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 degrees C but was almost inactive at 60 degrees C. E' stimulated the activity of the core enzyme at 60 degrees 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.
Naji
Souad
S
Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universität Regensburg, 93053 Regensburg, Germany.
Grünberg
Sebastian
S
Thomm
Michael
M
D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic / Q000235:genetics*
D019843:Archaeal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D012319:RNA Polymerase II