Sequeval D, Felenbok B
Institut de Génétique et Microbiologie, Université Paris-Sud XI, Centre Universitaire d'Orsay, France.
Mol Gen Genet. 1994 Jan;242(1):33-9. doi: 10.1007/BF00277345.
The transcription factor ALCR of the ethanol utilisation pathway in Aspergillus nidulans contains a zinc binuclear motif (CysX2CysX6CysX16CysX2CysX6Cys), within the DNA-binding domain located in the N-terminal region of the ALCR protein. Specific targets have been localised in the promoter of the alcR gene, involved in the autoregulation process, and in the promoter of the structural gene alcA (encoding alcohol dehydrogenase I), which is also under the control of ALCR. The DNA-binding domain has been expressed in-Escherichia coli as a GST-ALCR (7-58*) fusion protein and also obtained as an ALCR (7-58*) peptide. Both the ALCR fusion protein and the ALCR peptide are able to bind 65Zn(II) in vitro, if reduction of cysteines occurs prior to the addition of zinc. Competition experiments showed that Cd(II), Co(II) and Cu(II) are efficient competitors for the zinc binding sites. The ALCR DNA-binding domain was shown to contain 2 mol of tightly bound Zn(II) per mole of fusion protein. Removal of the intrinsic Zn(II) requires treatment with Chelex. This treatment abolishes the ability of the protein to bind to the targets of ALCR located in the alcA and alcR promoters. The apo-ALCR DNA-binding motif could be reconstituted with Zn(II) or Cd(II), restoring specific DNA binding to both types of targets. Thus a direct relationship was shown to exist between the zinc content of ALCR and its DNA-binding activity.
构巢曲霉乙醇利用途径中的转录因子ALCR在位于ALCR蛋白N端区域的DNA结合结构域内含有一个锌双核基序（CysX2CysX6CysX16CysX2CysX6Cys）。已在参与自调控过程的alcR基因启动子以及同样受ALCR调控的结构基因alcA（编码乙醇脱氢酶I）的启动子中定位了特定靶点。DNA结合结构域已在大肠杆菌中作为GST-ALCR（7-58*）融合蛋白表达，也作为ALCR（7-58*）肽获得。如果在添加锌之前半胱氨酸发生还原，那么ALCR融合蛋白和ALCR肽在体外均能够结合65Zn（II）。竞争实验表明，Cd（II）、Co（II）和Cu（II）是锌结合位点的有效竞争者。结果显示，每摩尔融合蛋白，ALCR DNA结合结构域含有2摩尔紧密结合的Zn（II）。去除内在的Zn（II）需要用螯合树脂处理。这种处理消除了蛋白质与位于alcA和alcR启动子中的ALCR靶点结合的能力。脱辅基ALCR DNA结合基序可用Zn（II）或Cd（II）重构，恢复与两种类型靶点的特异性DNA结合。因此，已表明ALCR的锌含量与其DNA结合活性之间存在直接关系。
