Paietta J V
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA.
Nucleic Acids Res. 1995 Mar 25;23(6):1044-9. doi: 10.1093/nar/23.6.1044.
The CYS3 positive regulator is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of sulfur-controlled structural genes in Neurospora crassa. An approach of modifying the dimerization specificity of the CYS3 leucine zipper was used to determine whether the in vivo regulatory function of CYS3 requires the formation of homodimeric or heterodimeric complexes. Two altered versions of CYS3 with coiled coil elecrostatic interactions favorable to heterodimerization showed restoration of wild-type CYS3 function only when simultaneously expressed in a delta cys-3 strain. In addition, constructs having the CYS3 leucine zipper swapped for that of the oncoprotein Jun or the CYS3 leucine zipper extended by a heptad repeat showed wild-type CYS3 function when transformed into a delta cys-3 strain. Gel mobility shift and immunoprecipitation assays were used to confirm the modified CYS3 proteins dimerization and DNA binding properties. The studies, which precluded wild-type CYS3 dimerization, indicate that in vivo CYS3 is fully functional as a homodimer since no interaction was required with other leucine zipper proteins to activate sulfur regulatory and structural gene expression. The results demonstrate the utility of leucine zipper modification to study the in vivo function of bZIP proteins.
CYS3正调控因子是一种碱性区域-亮氨酸拉链（bZIP）DNA结合蛋白，对粗糙脉孢菌中硫控制的结构基因的表达至关重要。采用一种改变CYS3亮氨酸拉链二聚化特异性的方法来确定CYS3在体内的调控功能是否需要形成同二聚体或异二聚体复合物。两个具有有利于异二聚化的卷曲螺旋静电相互作用的CYS3变体，只有在δcys-3菌株中同时表达时，才显示出野生型CYS3功能的恢复。此外，将CYS3亮氨酸拉链替换为癌蛋白Jun的亮氨酸拉链或通过七肽重复序列延伸CYS3亮氨酸拉链的构建体，在转化到δcys-3菌株中时显示出野生型CYS3功能。凝胶迁移率变动分析和免疫沉淀分析用于确认修饰后的CYS3蛋白的二聚化和DNA结合特性。这些排除了野生型CYS3二聚化的研究表明，在体内CYS3作为同二聚体具有完全功能，因为激活硫调控和结构基因表达不需要与其他亮氨酸拉链蛋白相互作用。结果证明了亮氨酸拉链修饰在研究bZIP蛋白体内功能方面的实用性。
