DNA binding and dimerization specificity and potential targets for the TCP protein family
DNA binding and dimerization specificity and potential targets for the TCP protein family
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DOI：
10.1046/j.1365-313x.2002.01294.x
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发表时间：
2002-05-01
期刊：
影响因子：
7.2
通讯作者：
Ohashi, Y
中科院分区：
文献类型：
--
作者：
Kosugi, S;Ohashi, Y
TCP结构域是一种植物特异性的DNA结合结构域，存在于多种物种的蛋白质中，包括cycloidea（cyc）和teosinte branched 1（tb 1）基因产物以及PCF 1和PCF 2蛋白质。为了了解这个结构域的蛋白质在转录调控中的作用，我们分析了DNA结合和二聚化特异性的TCP蛋白质家族使用水稻PCF蛋白，并进一步评估了TCP蛋白质的潜在目标。七个PCF成员，包括五个新分离的蛋白质，能够被分为两类，I和II，基于TCP域中的序列相似性。随机结合位点选择实验和电泳迁移率变动分析（EMSA）揭示了这两类的共识DNA结合序列是不同的，但重叠; GGNCCCAC为I类和GTGGNCCC为II类。玉米TB 1蛋白属于II类，与水稻II类蛋白具有相同的特异性，表明不同物种TCP结构域之间的结合特异性保守。酵母双杂交试验和EMSA表明，这些蛋白倾向于形成同源二聚体或异源二聚体之间的成员相同的类。我们在预测的拟南芥基因的5'侧翼序列中寻找共有结合序列，发现共有位点在基因组中以相当低的频率分布。我们进一步分析了八个启动子包含I类共识TCP网站。六个启动子的转录活性降低了TCP结合位点的突变，这与观察到的I类TCP位点可以赋予异源启动子反式激活功能是一致的。这些结果表明，这两类TCP蛋白在DNA结合特异性和转录调控方面是不同的。
The TCP domain is a plant-specific DNA binding domain found in proteins from a diverse array of species, including the cycloidea (cyc) and teosinte branched1 (tb1) gene products and the PCF1 and PCF2 proteins. To understand the role in transcriptional regulation of proteins with this domain, we have analysed the DNA binding and dimerization specificity of the TCP protein family using rice PCF proteins, and further evaluated potential targets for the TCP protein. The seven PCF members including five newly isolated proteins, were able to be grouped into two classes, I and II, based on sequence similarity in the TCP domain. Random binding site selection experiments and electrophoretic mobility shift assays (EMSAs) revealed the consensus DNA binding sequences of these two classes to be distinct but overlapping; GGNCCCAC for class I and GTGGNCCC for class II. The TB1 protein from maize, which belongs to class II, had the same specificity as the rice class II proteins, suggesting the conservation of binding specificity between TCP domains from different species. The yeast 2-hybrid assay and EMSA revealed that these proteins tend to form a homodimer or a heterodimer between members of the same class. We searched predicted 5' flanking sequences of Arabidopsis genes for the consensus binding sequences and found that the consensus sites are distributed in the genome at a considerably lower frequency. We further analysed eight promoters containing the class I consensus TCP sites. The transcriptional activities of six promoters were decreased by a mutation of the TCP binding site, which is consistent with the observation that the class I TCP site can confer transactivation function on a heterologous promoter. These results suggest that the two classes of TCP protein are distinct in DNA binding specificity and transcriptional regulation.