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.
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.
Monti
Maria C
MC
Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Hernández-Arriaga
Ana M
AM
Kamphuis
Monique B
MB
López-Villarejo
Juan
J
Heck
Albert J R
AJ
Boelens
Rolf
R
Díaz-Orejas
Ramón
R
van den Heuvel
Robert H H
RH
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C068354:Kis protein, Bacteria; D021122:Protein Subunits; C473072:kid toxin protein, E coli plasmid R1