The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure–function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the α subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the ΔH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.
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February 2009
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Conference Article|
January 20 2009
Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases
Michael Thomm;
Michael Thomm
1
1Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany
1To whom correspondence should be addressed (email michael.thomm@biologie.uni-regensburg.de)
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Christoph Reich;
Christoph Reich
1Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany
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Sebastian Grünberg;
Sebastian Grünberg
1Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany
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Souad Naji
Souad Naji
1Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany
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Publisher: Portland Press Ltd
Received:
August 14 2008
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem Soc Trans (2009) 37 (1): 18–22.
Article history
Received:
August 14 2008
Citation
Michael Thomm, Christoph Reich, Sebastian Grünberg, Souad Naji; Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases. Biochem Soc Trans 1 February 2009; 37 (1): 18–22. doi: https://doi.org/10.1042/BST0370018
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