Harmer T, Wu M, Schleif R
Biology Department, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):427-31. doi: 10.1073/pnas.98.2.427.
We applied two experiments useful in the study of ligand-regulated DNA binding proteins to AraC, the dimeric regulator of the Escherichia coli l-arabinose operon. In the absence of arabinose, AraC prefers to loop DNA by binding to two half-sites that are separated by 210 base pairs, and in the presence of arabinose it prefers to bind to adjacently located half-sites. The basis for this ligand-regulated shift in binding appears to result from a shift in the rigidity of the system, where rigidity both in AraC protein in the absence of arabinose, and in the DNA are required to generate the free energy differences that produce the binding preferences. Eliminating the dimerization domains and connecting the two DNA binding domains of AraC by a flexible peptide linker should provide a protein whose behavior mimics that of AraC when there is no interaction between its dimerization and DNA binding domains. The resulting protein bound to adjacent half-sites on the DNA, like AraC protein in the presence of arabinose. When the two double-stranded DNA half-sites were connected by 24 bases of single-stranded, flexible DNA, wild-type AraC protein bound to the DNA in the presence and absence of arabinose with equal affinity, showing that AraC modulates its DNA binding affinity in response to arabinose by shifting the relative positions of its DNA binding domains. These results are consistent with the light switch mechanism for the action of AraC, refine the model, and extend the range of experimental tests to which it has been subjected.
我们将两个在研究配体调节的DNA结合蛋白中有用的实验应用于阿拉伯糖操纵子二聚体调节蛋白AraC，该操纵子存在于大肠杆菌中。在没有阿拉伯糖的情况下，AraC倾向于通过结合两个相隔210个碱基对的半位点使DNA成环，而在有阿拉伯糖的情况下，它倾向于结合相邻的半位点。这种结合中配体调节的转变的基础似乎是由于系统刚性的转变，在没有阿拉伯糖时AraC蛋白以及DNA中的刚性都是产生结合偏好所需的自由能差异所必需的。去除二聚化结构域并用柔性肽接头连接AraC的两个DNA结合结构域，应该会产生一种蛋白质，其行为在其二聚化结构域和DNA结合结构域之间没有相互作用时模仿AraC的行为。产生的蛋白质与DNA上相邻的半位点结合，就像在有阿拉伯糖时的AraC蛋白一样。当两个双链DNA半位点通过24个单链柔性DNA碱基连接时，野生型AraC蛋白在有和没有阿拉伯糖的情况下以相同的亲和力结合到DNA上，表明AraC通过改变其DNA结合结构域的相对位置来响应阿拉伯糖调节其DNA结合亲和力。这些结果与AraC作用的光开关机制一致，完善了模型，并扩展了其经受的实验测试范围。
