Constitutive Mutations in the Escherichia coli AraC Protein
Constitutive Mutations in the Escherichia coli AraC Protein
复制标题
DOI：
10.1128/jb.01529-08
复制
发表时间：
2009-04-15
影响因子：
3.2
通讯作者：
Schleif, Robert
中科院分区：
文献类型：
--
作者：
Dirla, Stephanie;Chien, John Yeh-Heng;Schleif, Robert
大肠杆菌AraC蛋白抑制和诱导araBAD操纵子，以响应L-阿拉伯糖的存在或不存在。AraC基因的结构性突变不再需要L-阿拉伯糖的存在来将AraC从抑制状态转换到诱导状态。这些突变是通过它们的构成或对不可代谢的阿拉伯糖类似物D-岩藻糖的抗性而直接分离出来的。大多数结构性突变位于AraC的N端调控臂的相同残基内。然而，在二聚化结构域的核心发现了两个。正如AraC的光开关机制所预测的那样，结构性突变增加了N端手臂对胰酶或胰凝乳素消化的敏感性，表明这些突变削弱或破坏了AraC抑制所需的手臂结构。荧光、圆二色谱和半胱氨酸反应性测量表明，二聚化结构域核心的结构性突变导致对臂的支撑力减弱，降低了负阿拉伯糖臂结构的稳定性。这些突变还削弱了双螺旋束与二聚化结构域的β-桶亚域之间的相互作用，降低了β-桶的结构稳定性。
The Escherichia coli AraC protein represses and induces the araBAD operon in response to the absence or presence of L-arabinose. Constitutive mutations in the AraC gene no longer require the presence of L-arabinose to convert AraC from its repressing to its inducing state. Such mutations were isolated directly by virtue of their constitutivity or by their resistance to the nonmetabolizable arabinose analog, D-fucose. The majority of the constitutive mutations lie within the same residues of the N-terminal regulatory arm of AraC. Two, however, were found in the core of the dimerization domain. As predicted by the light switch mechanism of AraC, constitutive mutations increase the susceptibility of the N-terminal arms to digestion by trypsin or chymotrypsin, suggesting that these mutations weaken or disrupt the arm structure required for repression by AraC. Fluorescence, circular dichroism, and cysteine reactivity measurements show that the constitutive mutations in the core of the dimerization domain lead to a weakening of the support for the arms and reduce the stability of the minus-arabinose arm structure. These mutations also weaken the interaction between the two-helix bundle and the beta-barrel subdomains of the dimerization domain and reduce the structural stability of the beta-barrels.