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

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• 作者：Silvia Zorrilla ; Thierry Doan ; Carlos Alfonso ; Emmanuel Margeat ; Alvaro Ortega ; Germá ; n Rivas ; Sté ; phane Aymerich ; Catherine A. Royer ; Nathalie Declerck
• 刊名：Biophysical Journal
• 出版年：2007
• 出版时间：1 May 2007
• 年：2007
• 卷：92
• 期：9
• 页码：3215-3227
• 全文大小：356 K

文摘

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.