Nucleic Acids Research Advance Access originally published online on February 21, 2007
Nucleic Acids Research 2007 35(5):1737-1749; doi:10.1093/nar/gkm073
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Nucleic Acids Research, 2007, Vol. 35, No. 5 1737-1749
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Structural Biology |
Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers
1Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, 2Centro de Investigaciones Biológicas, Departamento de Microbiología Molecular, Ramiro de Maeztu 9, E-28040 Madrid, Spain and 3Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
*To whom correspondence should be addressed. Tel: +31 302536797; Fax: +31 302518219; Email: r.h.h.vandenheuvel{at}chem.uu.nl or robert.vandenheuvel{at}organon.com
Received October 17, 2006. Revised December 30, 2006. Accepted January 24, 2007.
The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors Present address: Robert H. H. van den Heuvel, Organon, P. O. Box 20, 5340 BH Oss, The Netherlands
Present address: Maria C. Monti, Diparimento di Scienze Farmaceutiche, Universita’ di Salerno, Via Ponte Don Mellilo, 84084, Fisciano, Salerno, Italy
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