CONSTRUCTION OF A SYNTHETIC GENE FOR THE METALLOREGULATORY PROTEIN MERR AND ANALYSIS OF REGIONALLY MUTATED PROTEINS OR TRANSCRIPTIONAL REGULATION

The transcriptional control protein MerR is a metalloregulatory switch , activating transcription of a mercury resistance operon in the prese nce 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 o f the MerR protein's function. Site-directed mutagenesis of clustered acidic residues within the central region of the MerR protein indicate d that these residues are important to the protein's ability to repres s transcription. Quadruple or sextuple mutations involving residues E8 3 and E84 and other nearby acidic residues result in a repression-defi cient (RD) phenotype. One of the mutant proteins was purified and show n by gel shift assay to retain binding to its operator DNA with an aff inity similar to wild-type protein, suggesting that transcriptional re pression does not correlate with MerR binding affinity. A small region of merR corresponding to residues 81-92 also was mutagenized in a sea rch for other RD mutants and for mutants displaying sufficient transcr iptional activation in the absence of mercuric ion to be classified as constitutive activation (CA) mutants. In this case, oligonucleotide-d irected randomization of the target region and a screening/selection p rotocol were employed. Sixteen different mutants with an RD phenotype were identified, as well as eight different mutants with a CA phenotyp e. A high frequency of S87C mutations is evident in the RD set of muta nts. 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 D NA site with an affinity similar to that of the wild-type protein. Che mical nuclease activity assays indicate that the nonmercurated S86C/A8 9V CA mutant has a DNA distortion activity identical to that of mercur ated wild-type MerR. A unique disulfide bond bridging this CA mutant's dimer interface was found and is proposed to constrain protein confor mation in a manner analogous to mercuric ion binding in the wild-type protein.