Inducer-modulated cooperative binding of the tetrameric CggR repressor to operator DNA.
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.
Zorrilla
Silvia
S
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.
Doan
Thierry
T
Alfonso
Carlos
C
Margeat
Emmanuel
E
Ortega
Alvaro
A
Rivas
Germán
G
Aymerich
Stéphane
S
Royer
Catherine A
CA
Declerck
Nathalie
N
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*
C473792:CggR protein, Bacillus subtilis; D004269:DNA, Bacterial; D005635:Fructosediphosphates; D012097:Repressor Proteins; C029063:fructose-1,6-diphosphate