Nagano Y, Furuhashi H, Inaba T, Sasaki Y
Graduate School of Agricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Nucleic Acids Res. 2001 Oct 15;29(20):4097-105. doi: 10.1093/nar/29.20.4097.
Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x(2)-H-x(11)-C-x(2)-C-x((4-5))-C-x(2)-C-x((3-7))-H-x(2)-H and C-x(2)-C-x((10-11))-C-x(3)-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with (65)ZnCl(2) showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression.
从豌豆中分离出了编码一种DNA结合蛋白的互补DNA，该蛋白命名为PLATZ1（富含AT序列和锌结合蛋白1）。该蛋白的氨基酸序列与从高等植物基因组序列预测的其他未表征蛋白的序列相似。然而，在植物界之外未发现旁系同源序列。这些旁系同源蛋白之间的多序列比对表明，几个半胱氨酸和组氨酸残基是不变的，这表明这些蛋白是一类新型的锌依赖性DNA结合蛋白，具有两个相距较远的区域，即C-x(2)-H-x(11)-C-x(2)-C-x((4-5))-C-x(2)-C-x((3-7))-H-x(2)-H和C-x(2)-C-x((10-11))-C-x(3)-C。在电泳迁移率变动分析中，锌螯合剂1,10-邻菲罗啉抑制了DNA结合，并且DNA结合需要两个相距较远的锌结合区域。用(65)ZnCl(2)进行的蛋白质印迹显示，两个区域对于锌结合活性都是必需的。PLATZ1蛋白非特异性地结合富含A/T的序列，包括豌豆GTP酶pra2和质体蓝素petE基因的上游区域。PLATZ1的表达抑制了那些含有由花椰菜花叶病毒（CaMV）35S90启动子驱动的、与富含A/T序列串联重复序列融合的荧光素酶基因编码序列的报告构建体的表达。这些结果表明，PLATZ1是一类新型的植物特异性锌依赖性DNA结合蛋白，负责富含A/T序列介导的转录抑制。
