Reaching out. Locating and lengthening the interdomain linker in AraC protein.
Reaching out. Locating and lengthening the interdomain linker in AraC protein.
复制标题
伸出手来。
DOI：
10.1006/jmbi.1994.1584
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发表时间：
1994
影响因子：
5.6
通讯作者：
Schleif,RF
中科院分区：
文献类型：
--
作者：
Eustance,RJ;Bustos,SA;Schleif,RF
开发了一种遗传方法来确定蛋白质中耐受插入和缺失的区域。这些区域有吸引力的候选区域是连接区。发现这样一个区域包括大肠杆菌调节蛋白AraC的第171到178位。独立的生化方法确定11到170个氨基酸残基是AraC的最小二聚化结构域，291个残基蛋白中178到286个氨基酸残基是最小的DNA结合区。因此，通过遗传和生化方法，确定了结构域之间的连接区包括171到177个氨基酸残基。使用具有比野生型更广泛的分离的AraC半位点的模板来检测改变的蛋白质的性质。结构域间连接区插入的AraC蛋白和由39个氨基酸残基连接区隔开的最小功能二聚化和DNA结合区的蛋白都能够结合在这样的DNA位点上并在其上发挥作用。在体外，具有较长连接区的蛋白质比野生型AraC蛋白更稳定地结合到由额外两个螺旋DNA所分隔的含有AraC半位点的DNA上。在活体启动子中，具有更广泛分离的AraC半位点的蛋白质可以比野生型AraC蛋白更好地激活转录。
A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions. Attractive candidates for these areas are linker regions. Such a region was found to include positions 171 to 178 in theEscherichia coliregulatory protein AraC. Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domain. Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177. The properties of altered proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case. Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site.In vitro, the proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA.In vivoon anarapromoter with the more widely separated AraC half-sites, the proteins could activate transcription much better than wild-type AraC.