Analysis of CYS3 regulator function in Neurospora crassa by modification of leucine zipper dimerization specificity.
Analysis of CYS3 regulator function in Neurospora crassa by modification of leucine zipper dimerization specificity.
复制标题
通过修饰亮氨酸拉链二聚化特异性分析粗糙脉孢菌中的 CYS3 调节功能。
DOI：
10.1093/nar/23.6.1044
复制
发表时间：
1995
影响因子：
14.9
通讯作者：
Paietta,JV
中科院分区：
文献类型：
--
作者：
Paietta,JV
CYS 3正调控蛋白是一种碱性亮氨酸拉链（bZIP）DNA结合蛋白，在粗糙脉孢菌硫调控结构基因的表达中起重要作用。修饰CYS 3亮氨酸拉链的二聚化特异性的方法用于确定CYS 3的体内调节功能是否需要形成同源二聚体或异源二聚体复合物。两种具有卷曲螺旋静电相互作用有利于异源二聚化的CYS 3的改变版本仅当在δcys-3菌株中同时表达时才显示野生型CYS 3功能的恢复。此外，将CYS 3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延伸的CYS 3亮氨酸拉链的构建体在转化为δcys-3菌株时显示野生型CYS 3功能。凝胶迁移率变化和免疫沉淀试验用于确认修饰的CYS 3蛋白二聚化和DNA结合性质。排除了野生型CYS 3二聚化的研究表明，体内CYS 3作为同源二聚体是完全功能性的，因为不需要与其他亮氨酸拉链蛋白相互作用来激活硫调节和结构基因表达。结果表明亮氨酸拉链修饰可用于研究bZIP蛋白的体内功能。
ABSTRACTThe CYS3 positive regulator is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of sulfur-controlled structural genes inNeurospora crassa. An approach of modifying the dimerization specificity of the CYS3 leucine zipper was used to determine whether thein vivoregulatory function of CYS3 requires the formation of homodimeric or heterodimeric complexes. Two altered versions of CYS3 with coiled coil electrostatic interactions favorable to heterodimerization showed restoration of wild-type CYS3 function only when simultaneously expressed in a δcys-3strain. 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 whentransformed into a δcys-3strain. Gel mobility shift and immunoprecipita lion assays were used to confirm the modified CYS3 proteins dimerization and DNA binding properties. The studies, which precluded wild-type CYS3 dimerization, indicate thatin vivoCYS3 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 thein vivofunction of bZIP proteins.