DETERMINANTS OF SPECIFICITY IN THE LACTOSE REPRESSOR

乳糖抑制物与乳糖操纵子的相互作用，以及 一般情况下，DNA将继续通过遗传和 生化手段。特异性改变的阻遏突变体 与改变的(0-c)算子紧密结合将通过一种新的对偶得到 使用对称野型和0-c运算符的选择方案。 将对LACI中改变的特异性突变进行测序，以绘制出 决定DNA结合的初级蛋白质结构的区域。至 螺旋-转角-螺旋模型对蛋白质识别的检验预测 DNA，将在lac阻遏物中进行特定的氨基酸替换 利用人工合成的DNA片段对LacI进行定向诱变。部分 基因头部区域的部分将被化学合成以 引入碱基对变化，从而产生唯一的限制性内切酶切点。小的 然后，基因片段将被移除，并被双链体取代 携带已知的突变。每个突变抑制子的稳定性，以及 由于其与野生型操纵子的亲和力将在体内和在 体外培养。将检查来自选定抑制物的氨基末端头饰 用于体外与野生型突变操纵子的特异性结合。

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.