Comess K M, Shewchuk L M, Ivanetich K, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1994 Apr 12;33(14):4175-86. doi: 10.1021/bi00180a010.
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.
转录控制蛋白MerR是一种金属调节开关，在汞离子存在时激活汞抗性操纵子的转录，而在其不存在时抑制转录。我们在此报告合成merR基因和单拷贝merT'-lacZ融合报告基因的构建及用于MerR蛋白功能诱变分析的情况。对MerR蛋白中心区域内成簇酸性残基进行定点诱变表明，这些残基对蛋白抑制转录的能力很重要。涉及残基E83和E84以及其他附近酸性残基的四重或六重突变导致抑制缺陷（RD）表型。其中一种突变蛋白被纯化，凝胶迁移分析表明其与操纵子DNA的结合亲和力与野生型蛋白相似，这表明转录抑制与MerR的结合亲和力无关。还对merR中对应于残基81 - 92的一个小区域进行了诱变，以寻找其他RD突变体以及在无汞离子时显示出足够转录激活从而可归类为组成型激活（CA）突变体的突变体。在这种情况下，采用了靶区域的寡核苷酸定向随机化以及筛选/选择方案。鉴定出16种不同的具有RD表型的突变体以及8种不同的具有CA表型的突变体。在RD突变体组中明显存在高频率的S87C突变。CA突变体中S86C和A89V突变的发生率很高。纯化了CA双突变体S86C/A89V，发现其与DNA位点的结合亲和力与野生型蛋白相似。化学核酸酶活性分析表明，未结合汞的S86C/A89V CA突变体具有与结合汞的野生型MerR相同的DNA扭曲活性。发现了一个独特的二硫键桥接该CA突变体的二聚体界面，并提出其以类似于野生型蛋白中汞离子结合的方式限制蛋白质构象。
