Construction of a synthetic gene for the metalloregulatory protein MerR and analysis of regionally mutated proteins for transcriptional regulation.
The transcriptional control protein MerR is a metalloregulatory switch, activating transcription of a mercury resistance operon in the presence of mercuric ions and repressing transcription in their absence. We report here the construction and utilization of a synthetic merR gene and a single-copy merT'-lacZ fusion reporter for mutagenic analysis of the MerR protein's function. Site-directed mutagenesis of clustered acidic residues within the central region of the MerR protein indicated that these residues are important to the protein's ability to repress transcription. Quadruple or sextuple mutations involving residues E83 and E84 and other nearby acidic residues result in a repression-deficient (RD) phenotype. One of the mutant proteins was purified and shown by gel shift assay to retain binding to its operator DNA with an affinity similar to wild-type protein, suggesting that transcriptional repression does not correlate with MerR binding affinity. A small region of merR corresponding to residues 81-92 also was mutagenized in a search for other RD mutants and for mutants displaying sufficient transcriptional activation in the absence of mercuric ion to be classified as constitutive activation (CA) mutants. In this case, oligonucleotide-directed randomization of the target region and a screening/selection protocol were employed. Sixteen different mutants with an RD phenotype were identified, as well as eight different mutants with a CA phenotype. A high frequency of S87C mutations is evident in the RD set of mutants. The CA mutants have a high incidence of S86C and A89V mutations. The CA double mutant S86C/A89V was purified and found to bind to its DNA site with an affinity similar to that of the wild-type protein. Chemical nuclease activity assays indicate that the nonmercurated S86C/A89V CA mutant has a DNA distortion activity identical to that of mercurated wild-type MerR. A unique disulfide bond bridging this CA mutant's dimer interface was found and is proposed to constrain protein conformation in a manner analogous to mercuric ion binding in the wild-type protein.
Comess
K M
KM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Shewchuk
L M
LM
Ivanetich
K
K
Walsh
C T
CT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010618:Phenanthrolines / Q000737:chemistry; D011994:Recombinant Proteins; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C051124:MerR protein, Bacteria; D010618:Phenanthrolines; D011994:Recombinant Proteins; C038556:bis(1,10-phenanthroline)copper(1+) ion