title: Second monomer binding is the rate-limiting step in the formation of the dimeric PhoP-DNA complex.
abstract: PhoP, the response regulator of the PhoP/PhoQ system, regulates Mg(2+) homeostasis in Salmonella typhimurium. Dimerization of PhoP on the DNA is necessary for its regulatory function, and PhoP regulates the expression of genes in a phosphorylation-dependent manner. Higher PhoP concentrations, however, can activate PhoP and substitute for phosphorylation-dependent gene regulation. Activation of PhoP by phosphorylation is explained by self-assembly of phosphorylated PhoP (PhoP-p) in solution and binding of the PhoP-p dimer to the promoter. To understand the mechanism of PhoP dimerization on the DNA, we examined the interactions of PhoP with double-stranded DNAs containing the canonical PhoP box (PB). We present results from multiple biophysical methods, demonstrating that PhoP is a monomer in solution over a range of concentrations and binds to PB in a stepwise manner with a second PhoP molecule binding weakly. The affinity for the binding of the first PhoP molecule to PB is more than ∼17-fold higher than the affinity of the second PhoP monomer for PB. Kinetic analyses of PhoP binding reveal that the on rate of the second PhoP monomer binding is the rate-limiting step during the formation of the (PhoP)(2)-DNA complex. Results show that a moderate increase in PhoP concentration can promote dimerization of PhoP on the DNA, which otherwise could be achieved by PhoP-p at much lower protein concentrations. Detailed analyses of PhoP-DNA interactions have revealed the existence of a kinetic barrier that is the key for specificity in the formation of the productive (PhoP)(2)-DNA complex.
authors.affiliation: Council of Scientific and Industrial Research, Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India.
authors.forename: Vijay
authors.initials: V
authors.lastname: Singh
authors.forename: Mary Krishna
authors.initials: MK
authors.lastname: Ekka
authors.forename: Sangaralingam
authors.initials: S
authors.lastname: Kumaran
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology*; D001703:Biophysics / Q000379:methods; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry*; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D012486:Salmonella typhimurium / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; C061064:PhoP protein, Bacteria; D004247:DNA; D008274:Magnesium