DETERMINANTS OF SPECIFICITY IN THE LACTOSE REPRESSOR

乳糖阻遏物与乳糖操纵子的相互作用，以及 一般来说，DNA将继续通过遗传和 生化手段。 特异性改变的阻遏突变体， 紧结合到改变的（0-c）算子将获得一个新的对偶 使用对称野生型和0-c算子的选择方案。 将对lacI中特异性改变的突变进行测序，以绘制出 决定DNA结合的蛋白质一级结构区域。 到 测试预测的螺旋-转角-螺旋模型的蛋白质识别 DNA，在乳糖阻遏物中进行特定的氨基酸取代， 用合成的DNA片段对lacI进行定向诱变。 部分 基因的头部区域将被化学合成， 引入碱基对变化，产生独特限制性位点。 小 基因片段将被移除， 携带已知的突变 每个突变阻遏物的稳定性，以及 因为其对野生型操纵子的亲和力将在体内和体内测定， 体外 将检查来自选定阻遏物的氨基末端头部片段 用于在体外特异性结合野生型突变体操纵子。

The interaction of the lactose repressor with the lactose operator, and with DNA in general, will continue to be analyzed by genetic and biochemical means. Repressor mutants with specificities altered so as to bind tightly to altered (0-c) operators will be obtained by a novel dual selection scheme using symmetric wild-type and 0-c operators. Altered-specificity mutations in lacI will be sequenced to map out the regions of the primary protein structure which determine DNA binding. To test predictions of the helix-turn-helix model for protein recognition of DNA, specific amino acid substitutions in the lac repressor will be made using directed mutagenesis of lacI with synthetic DNA fragments. Portions of the headpiece region of the gene will be chemically synthesized to introduce base pair changes that create unique restriction sites. Small segments of the gene will then be removed and replaced with duplexes carrying known mutations. The stability of each mutant repressor, as well as its affinity for wild-type operator will be assayed in vivo and in vitro. Amino-terminal headpieces from selected repressors will be examined for specific binding to wild-type mutant operators in vitro.