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Inducer-Modulated Cooperative Binding of the Tetrameric CggR Repressor to Operator DNA

Silvia Zorrilla ^*, Thierry Doan , Carlos Alfonso , Emmanuel Margeat ^*, Alvaro Ortega , Germán Rivas , Stéphane Aymerich , Catherine A. Royer ^* and Nathalie Declerck ^* 

^* 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; Microbiologie et Génétique Moléculaire, INRA, UMR 1238, and Centre National de la Recherche Scientifique, UMR 2585, Institut National Agronomique Paris-Grignon, Thiverval-Grignon, France; Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu, Madrid, Spain; and Departamento de Química Física, Facultad de Química, Universidad de Murcia, Murcia, Spain

Correspondence: Address reprint requests to N. Declerck, Tel.: 33-4-67-41-79-11; E-mail: nathalie{at}cbs.cnrs.fr.

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 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.

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