Transcriptional switching by the metalloregulatory MerR protein: initial characterization of DNA and mercury (II) binding activities.
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 10(4) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Shewchuk
L M
LM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Verdine
G L
GL
Walsh
C T
CT
D000478:Alkylation; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D005786:Gene Expression Regulation; D007460:Iodoacetamide / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008628:Mercury / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism*; D008745:Methylation; D009154:Mutation; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013816:Thermodynamics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008024:Ligands; C051124:MerR protein, Bacteria; D008667:Metalloproteins; D004228:Dithionitrobenzoic Acid; D003850:Deoxyribonuclease I; D008628:Mercury; D003545:Cysteine; D007460:Iodoacetamide