Recombinant Thermus aquaticus RNA polymerase for structural studies

Journal of Molecular Biology
Konstantin KuznedelovK Severinov

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 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 ...Continue Reading

References

Mar 1, 1991·Acta Crystallographica. Section A, Foundations of Crystallography·T A JonesM Kjeldgaard
Jan 1, 1989·Molecular & General Genetics : MGG·R S JokerstA L Greenleaf
Mar 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·D SweetserR A Young
May 9, 1995·Proceedings of the National Academy of Sciences of the United States of America·K SeverinovS A Darst
May 23, 1995·Proceedings of the National Academy of Sciences of the United States of America·H TangR H Ebright
May 13, 1997·Proceedings of the National Academy of Sciences of the United States of America·P D AdamsA T Brünger
Sep 28, 1999·Cell·J FuR D Kornberg
Aug 1, 2000·Science·N KorzhevaS A Darst
Feb 7, 2001·Proceedings of the National Academy of Sciences of the United States of America·L MinakhinK Severinov
Apr 12, 2001·Current Opinion in Structural Biology·S A Darst
Feb 13, 2002·Current Opinion in Structural Biology·Patrick Cramer
Mar 21, 2002·Proceedings of the National Academy of Sciences of the United States of America·Seth A DarstWilly Wriggers
May 10, 2002·Nature·Dmitry G VassylyevShigeyuki Yokoyama
Aug 31, 2002·Science·Brian BenoffRichard H Ebright
Jan 15, 1962·Proceedings of the National Academy of Sciences of the United States of America·M CHAMBERLIN, P BERG
Jan 10, 2004·Methods in Enzymology·Konstantin KuznedelovKonstantin Severinov
Jan 10, 2004·Methods in Enzymology·Arkadv MustaevAlex Goldfarb
Jan 1, 1997·Methods in Enzymology·Zbyszek Otwinowski, Wladek Minor

❮ Previous
Next ❯

Citations

Jul 1, 2011·Journal of the American Chemical Society·Sergey V ProninSergey A Kozmin
Oct 27, 2009·Proceedings of the National Academy of Sciences of the United States of America·Nataliya MiropolskayaAndrey Kulbachinskiy
Jun 11, 2010·Proceedings of the National Academy of Sciences of the United States of America·Yulia Yuzenkova, Nikolay Zenkin
Dec 3, 2011·Proceedings of the National Academy of Sciences of the United States of America·Kelly-Anne F TwistSeth A Darst
Jun 25, 2008·The Journal of Biological Chemistry·Nataliya BarinovaAndrey Kulbachinskiy
Jan 27, 2011·Nucleic Acids Research·Mohammad RoghanianNikolay Zenkin
Jan 13, 2012·Nucleic Acids Research·Zhanna BerdygulovaLeonid Minakhin
Nov 16, 2013·Nucleic Acids Research·Han-Wen ChangVasily M Studitsky
Oct 4, 2013·Nucleic Acids Research·Nataliya MiropolskayaAndrey Kulbachinskiy
May 13, 2010·BMC Biology·Yulia YuzenkovaNikolay Zenkin
May 15, 2015·Biomolecules·Katsuhiko S Murakami
Nov 10, 2009·Journal of Molecular Biology·William J Lane, Seth A Darst
Feb 22, 2008·Protein Expression and Purification·Xiao Yang, Peter J Lewis
Nov 6, 2010·Journal of Molecular Biology·Zhanna BerdygulovaLeonid Minakhin
Jun 14, 2017·Proceedings of the National Academy of Sciences of the United States of America·Marina N VassylyevaDmitry G Vassylyev
Dec 5, 2008·Biochemistry·Jay D Gralla, Yi-Xin Huo
Oct 25, 2008·Methods : a Companion to Methods in Enzymology·Irina Artsimovitch, Tina M Henkin

❮ Previous
Next ❯

Related Concepts

Related Feeds

CRISPRi Screens for Antibiotics

The CRISPR-Cas system is a gene editing technique that can be used for high-throughput genome-wide screens to identify modes of actions of novel antibiotics. Here is the latest research.