Transcriptional switching by the metalloregulatory MerR protein: initial characterization of DNA and mercury(II) binding activities

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Abstract
The MerR protein from the Tn501 mercury resistance operon is a metalloregulatory transcriptional switch, converting from repressor to activator on binding of Hg(II). We have determined via binding studies with 203Hg(II) that a single Hg(II) atom binds to the MerR dimer (32 kDa) with a half-saturation concentration of 10-7 M in the presence of up to 10-3 M exogenous thiols. This 104 selective binding is specific for the binding of Hg(II) and corresponds to concentrations of metal that induce mercury (II) resistance in vivo. Kd values for MerR binding, in the absence and presence of Hg(II), to a 305 bp DNA fragment containing the 18 bp dyad symmetry element, DS1, located at -35 to -10 upstream of the mer structural genes, were determined by a gel shift assay. A Kd of 10-10 M for free MerR and 10-11 M for Hg(II)-MerR complexes was revealed. Measurements of koff values, by this assay, show equally long-lived complexes of MerR-DNA (51-min half-life) and Hg(II)-MerR-DNA (49-min half-life), suggesting that Hg(II) accelerates MerR binding to DNA rather than influencing the dissociation rate of the protein-DNA-complex. In contrast, 203Hg(II) studies reveal that mercuric ions rapidly dissociate and associate with MerR-DNA complexes. Extensive footprinting studies by DNase I, methylation protection, and hydroxyl radicals indicate MerR stays bound to DS1 even on addition of Hg(II) and shares no interaction in vitro with a second dyad symmetry element, DS2, centered at -79/-80. Studies with DTNB and pHMB titration of protein thiols and alkylation studies with iodo[14C]acetamide, in the presence and absence of stoichiometrically bound Hg(II), allow initial assessment of role for Cys-82, -115, -117 and -126 as potential ligands for Hg(II). A tryptic fragment of 1-120 amino acids (or 1-121 aa) still dimerizes and binds specifically to mer DNA but has lost 203Hg(II) binding capacity.