Published online 8 December 2004
Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved
DNA footprinting and biophysical characterization of the controller protein C.AhdI suggests the basis of a genetic switch
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth PO1 2DT, UK
* To whom correspondence should be addressed. Tel: +44 23292 842678; Fax: +44 23292 842030; Email: geoff.kneale{at}port.ac.uk
Received October 18, 2004; Revised and Accepted November 12, 2004
We have cloned and expressed the ahdIC gene of the AhdI restriction-modification system and have purified the resulting controller (C) protein to homogeneity. The protein sequence shows a HTH motif typical of that found in many transcriptional regulators. C.AhdI is found to form a homodimer of 16.7 kDa; sedimentation equilibrium experiments show that the dimer dissociates into monomers at low concentration, with a dissociation constant of 2.5 µM. DNase I and Exo III footprinting were used to determine the C.AhdI DNA-binding site, which is found 
30 bp upstream of the ahdIC operon. The intact homodimer binds cooperatively to a 35 bp fragment of DNA containing the C-protein binding site with a dissociation constant of 5–6 nM, as judged both by gel retardation analysis and by surface plasmon resonance, although in practice the affinity for DNA is dominated by protein dimerization as DNA binding by the monomer is negligible. The location of the C-operator upstream of both ahdIC and ahdIR suggests that C.AhdI may act as a positive regulator of the expression of both genes, and could act as a molecular switch that is critically dependent on the Kd for the monomer–dimer equilibrium. Moreover, the structure and location of the C.AhdI binding site with respect to the putative –35 box preceding the C-gene suggests a possible mechanism for autoregulation of C.AhdI expression.
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