title: Mutagenesis of the cysteines in the metalloregulatory protein MerR indicates that a metal-bridged dimer activates transcription.
abstract: Bacterial resistance to mercury(II) compounds is controlled by the metalloregulatory MerR protein, a transcriptional repressor and a mercuric ion dependent activator of the mer operon. Site-directed mutagenesis of all four cysteine residues in the Tn501 MerR protein has led to the specific replacement of C82, C115, and C117 with alanine and of C126 with serine. Mutation of C82 and C126 abolishes transcriptional activation in vivo while mutation of C115 and C117 leads to a slight increase and dramatic decrease in transcriptional activation, respectively. All four mutants are competent, to varying degrees, to repress mer transcription. Characterization of the four purified mutant proteins in vitro demonstrates that only the C126S MerR mutant is most notably deficient in stoichiometric Hg(II) binding. All four mutant proteins possess similar DNA binding properties, and the C82 mutant is most affected in the ability to form stable dimers. Given an observed stoichiometry of one Hg(II) per MerR dimer, it is likely that the transcriptionally activating MerR species is a metal-bridged dimer. It is most likely that one C126 per subunit provides high-avidity bidentate ligation to Hg(II), but it remains possible that C82 may be a secondary Hg(II) ligand (e.g., in a tetracoordinate thiol ligation array).
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: L M
authors.initials: LM
authors.lastname: Shewchuk
authors.forename: G L
authors.initials: GL
authors.lastname: Verdine
authors.forename: H
authors.initials: H
authors.lastname: Nash
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003545:Cysteine; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics*; D004352:Drug Resistance, Microbial; D005786:Gene Expression Regulation; D005796:Genes; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008628:Mercury / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009876:Operon; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C051124:MerR protein, Bacteria; D008628:Mercury; D003545:Cysteine