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Phosphorylation-induced dimerization of the FixJ receiver domain
S Da Re1, J Schumacher, P Rousseau
1Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215 INRA-CNRS, Chemin de Borde Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.
Molecular Microbiology
|November 17, 1999
Summary
The transcriptional activator FixJ in Sinorhizobium meliloti dimerizes upon phosphorylation, a process influenced by its domains. This dimerization is crucial for regulating nitrogen fixation by enabling DNA binding.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The transcriptional activator FixJ regulates nitrogen fixation in Sinorhizobium meliloti.
- FixJ belongs to the 'two-component' system family of transcriptional regulators.
Purpose of the Study:
- To investigate the mechanism of FixJ activation, focusing on phosphorylation-induced dimerization.
- To elucidate the role of FixJ domains in its regulatory activity and DNA binding.
Main Methods:
- Gel permeation chromatography and equilibrium sedimentation analysis to study protein dimerization.
- Chemical phosphorylation assays to determine reaction kinetics.
- Alanine scanning mutagenesis to identify key residues in the dimerization interface.
Main Results:
- Phosphorylation induces FixJ dimerization, an intrinsic property of its receiver domain (FixJN).
- The FixJ C-terminal domain (FixJC) inhibits FixJN phosphorylation, while FixJN inhibits FixJC activity, indicating domain interaction.
- Specific residues (Val-91 and Lys-95) in helix alpha4 of FixJN are critical for dimerization.
- Dimerization is essential for high-affinity binding of FixJ to the fixK promoter DNA.
Conclusions:
- FixJ activity is modulated by domain interactions and phosphorylation-induced dimerization.
- Disruption of the FixJN-FixJC interface and dimerization are key steps in activating FixJ for nitrogen fixation regulation.