Ramos J L, Michan C, Rojo F, Dwyer D, Timmis K
Estación Experimental del Zaidin, C.S.I.C., Granada, Spain.
J Mol Biol. 1990 Jan 20;211(2):373-82. doi: 10.1016/0022-2836(90)90358-S.
This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein. The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative beta-pleated domain. These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K's values fivefold to 60-fold lower than those of the wild-type XylS protein. They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring. Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative alpha-helix-rich domain. Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator. These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure. The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate. Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.
本研究报告了对基因表达正调控因子与其效应分子之间相互作用的遗传分析。TOL 质粒间位裂解途径操纵子的转录受到 XylS 蛋白正调控因子的特异性刺激，这要么是通过苯甲酸效应物激活该调控因子，要么是通过其过量产生来实现。已对一个表现出操纵子启动子组成型表达的 xylS 突变体以及六个编码改变的 XylS 蛋白的突变体进行了表征，这些改变的 XylS 蛋白将苯甲酸类似物识别为效应物，而这些苯甲酸类似物对 XylS 野生型蛋白而言并非效应物。两个突变调控因子中的变化位于蛋白质的 N 末端，在一个假定的β折叠结构域内。这些突变蛋白对正常苯甲酸效应物的亲和力显著增加，其解离常数（K 值）比野生型 XylS 蛋白低 5 倍至 60 倍。它们还被在芳香环的 2、3 和 4 位具有某些取代基的新效应物激活。另外两个突变蛋白识别在芳香环的 4 和 5 位具有取代基的新效应物，并且在其 C 末端的一个假定富含α螺旋的结构域内含有突变。另外三个突变，其中一个导致从 Pm 组成型表达，每个突变都导致调控因子中心区域的氨基酸变化。这些发现表明但未证明 XylS 蛋白的效应物结合口袋可能由其一级结构的两个或更多不连续片段组成。XylS 蛋白与大肠杆菌的 AraC 蛋白具有同源性，AraC 蛋白在被阿拉伯糖或苯甲酸激活时会刺激 ara 启动子的转录。本研究中表征的影响 XylS 蛋白效应物激活作用的突变均位于与 AraC 蛋白相应比对序列具有高度同源性的区域。
