Recombinant Thermus aquaticus RNA Polymerase for Structural Studies

Konstantin Kuznedelov, Valerie Lamour, Georgia Patikoglou, Mark Chlenov, Seth A. Darst, Konstantin Severinov

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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 β′ 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 Taqβ′NCD deletion mutants generated in this study showed that the β′NCD is important for the efficient binding of the σ subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1 Å resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the β-flap, further defining the range of conformations accessible to this flexible structural element.

Original languageEnglish (US)
Pages (from-to)110-121
Number of pages12
JournalJournal of molecular biology
Volume359
Issue number1
DOIs
StatePublished - May 26 2006

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • RNA polymerase
  • Thermus aquaticus
  • crystal structure

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