de Pater S, Greco V, Pham K, Memelink J, Kijne J
Center for Phytotechnology, Leiden University, The Netherlands.
Nucleic Acids Res. 1996 Dec 1;24(23):4624-31. doi: 10.1093/nar/24.23.4624.
The C2-H2 zinc-finger is a widely occurring DNA binding motif, usually present as tandem repeats. The majority of C2-H2 zinc-finger proteins that have been studied are derived from animals. Here, we characterize a member of a distinct class of plant C2-H2 zinc-finger proteins in detail. A cDNA clone encoding a DNA binding protein from Arabidopsis was isolated by SouthWestern screening. The protein, termed ZAP1 (Zinc-dependent Activator Protein-1), is encoded by a single copy gene, which is expressed to similar levels in root and flower, to a somewhat lower level in stem and to low levels in leaf and siliques. The optimal binding site was determined by random binding site selection, and the consensus sequence found is CGTTGACCGAG. The homology between ZAP1 and other DNA binding proteins is restricted to a repeated region of a stretch of 24 highly conserved amino acids followed by a zinc-finger motif (C-X4-C-X22-23-H-X1-H). The C-terminal zinc-finger region is essential for DNA binding, whereas deletion of the N-terminal one resulted in 2.5-fold reduced binding affinity. Binding of ZAP1 to DNA was abolished by metal-chelating agents. The activation domain as determined in yeast is adjacent to and possibly overlapping with the DNA binding domain. Particle bombardment experiments with plant cells showed that ZAP1 increases expression of a gusA reporter gene that is under control of ZAP1 binding sites. We conclude that ZAP1 is a plant transcriptional activator with a C2-H2 zinc-finger DNA binding domain.
C2-H2锌指是一种广泛存在的DNA结合基序，通常以串联重复的形式出现。大多数已研究的C2-H2锌指蛋白来源于动物。在此，我们详细描述了一类独特的植物C2-H2锌指蛋白中的一个成员。通过SouthWestern筛选分离出一个编码拟南芥DNA结合蛋白的cDNA克隆。该蛋白称为ZAP1（锌依赖性激活蛋白-1），由单拷贝基因编码，在根和花中的表达水平相似，在茎中的表达水平略低，在叶和角果中的表达水平较低。通过随机结合位点选择确定了最佳结合位点，发现的共有序列为CGTTGACCGAG。ZAP1与其他DNA结合蛋白之间的同源性仅限于一段由24个高度保守氨基酸组成的重复区域，其后是一个锌指基序（C-X4-C-X22-23-H-X1-H）。C末端锌指区域对于DNA结合至关重要，而N末端的缺失导致结合亲和力降低2.5倍。金属螯合剂可消除ZAP1与DNA的结合。在酵母中确定的激活结构域与DNA结合结构域相邻且可能重叠。对植物细胞进行的粒子轰击实验表明，ZAP1可增加受ZAP1结合位点控制的gusA报告基因的表达。我们得出结论，ZAP1是一种具有C2-H2锌指DNA结合结构域的植物转录激活因子。
