2gho
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Recombinant Thermus aquaticus RNA polymerase for Structural Studies
OverviewOverview
Advances in the structural biology of bacterial transcription have come, from studies of RNA polymerases (RNAPs) from the thermophilic eubacteria, Thermus aquaticus (Taq) and Thermus thermophilus (Tth). These structural, studies have been limited by the fact that only endogenous Taq or Tth, RNAP, laboriously purified from large quantities of Taq or Tth cell paste, and offering few options for genetic modification, is suitable for, structural studies. Recombinant systems for the preparation of Taq RNAP by, co-overexpression and assembly in the heterologous host, Escherichia coli, have been described, but these did not yield enzyme suitable for, crystallographic studies. Here we describe recombinant systems for the, preparation of Taq RNAP harboring full or partial deletions of the Taq, beta' non-conserved domain (NCD), yielding enzyme suitable for, crystallographic studies. This opens the way for structural studies of, genetically manipulated enzymes, allowing the preparation of more, crystallizable enzymes and facilitating detailed structure/function, analysis. Characterization of the Taqbeta'NCD deletion mutants generated, in this study showed that the beta'NCD is important for the efficient, binding of the sigma subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1Angstroms resolution) of one of the, recombinant mutants revealed a previously unobserved conformation of the, beta-flap, further defining the range of conformations accessible to this, flexible structural element.
About this StructureAbout this Structure
2GHO is a Protein complex structure of sequences from Thermus aquaticus. Active as DNA-directed RNA polymerase, with EC number 2.7.7.6 Full crystallographic information is available from OCA.
ReferenceReference
Recombinant Thermus aquaticus RNA polymerase for structural studies., Kuznedelov K, Lamour V, Patikoglou G, Chlenov M, Darst SA, Severinov K, J Mol Biol. 2006 May 26;359(1):110-21. Epub 2006 Mar 23. PMID:16618493
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