title: Inducer-modulated cooperative binding of the tetrameric CggR repressor to operator DNA.
abstract: The central glycolytic genes repressor (CggR) controls the transcription of the gapA operon encoding five key glycolytic enzymes in Bacillus subtilis. CggR recognizes a unique DNA target sequence comprising two direct repeats and fructose-1,6-bisphosphate (FBP) is the inducer that negatively controls this interaction. We present here analytical ultracentrifugation and fluorescence anisotropy experiments that demonstrate that CggR binds as a tetramer to the full-length operator DNA in a highly cooperative manner. We also show that CggR binds as a dimer to each direct repeat, the affinity being approximately 100-fold higher for the 3' repeat. In addition, our studies reveal a bimodal effect of FBP on the repressor/operator interaction. At micromolar concentrations, FBP leads to a change in the conformational dynamics of the complex. In the millimolar range, without altering the stoichiometry, FBP leads to a drastic reduction in the affinity and cooperativity of the complex. This bimodal response suggests the existence of two sugar-binding sites in the repressor, a high affinity site at which FBP acts as a structural co-factor and a low affinity site underlying the molecular mechanism of gapA induction.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 554; and Université Montpellier, Centre National de la Recherche Scientifique, UMR 5048, Centre de Biochimie Structurale, Montpellier, France.
authors.forename: Silvia
authors.initials: S
authors.lastname: Zorrilla
authors.forename: Thierry
authors.initials: T
authors.lastname: Doan
authors.forename: Carlos
authors.initials: C
authors.lastname: Alfonso
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Margeat
authors.forename: Alvaro
authors.initials: A
authors.lastname: Ortega
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.forename: Stéphane
authors.initials: S
authors.lastname: Aymerich
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Royer
authors.forename: Nathalie
authors.initials: N
authors.lastname: Declerck
mesh_terms: D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry*; D005635:Fructosediphosphates / Q000737:chemistry*; D007700:Kinetics; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry*
chemical_list: C473792:CggR protein, Bacillus subtilis; D004269:DNA, Bacterial; D005635:Fructosediphosphates; D012097:Repressor Proteins; C029063:fructose-1,6-diphosphate