Takatsuji H, Matsumoto T
Laboratory of Developmental Biology, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan.
J Biol Chem. 1996 Sep 20;271(38):23368-73. doi: 10.1074/jbc.271.38.23368.
The EPF family is a group of transcription factors containing canonical Cys2/His2-type zinc finger motifs that were first discovered in plants. These zinc finger proteins are characterized by two zinc fingers that are separated by spacers of various lengths, which are much longer than typical spacers (HC-link) in cluster-type zinc finger proteins. We describe here direct evidence that the two zinc fingers make contact with two tandemly repeated AGT core sequences that are separated by about 13 base pairs, by contrast to the cluster-type zinc finger proteins that bind to contiguous triplet sequences. DNA binding affinities were sensitive to the spaces between the core sequences, and the sensitivity to the spacing was greatly affected by the DNA sequence between the core sequences, with GC-rich sequences endowing much higher specificity than AT-rich sequences. Among the members of the EPF family, EPF1 was less sensitive to the spacing than EPF2-5. These results suggest that EPFs recognize their cognate target DNAs not only by the sequence of the core sites but also by the spacing between the core sites and, moreover, that different members in the EPF family distinguish their specific target genes by reference to these two parameters. This represents a unique type of target-sequence recognition among Cys2/His2-type zinc finger transcription factors. In addition, site-directed mutagenesis studies demonstrated that the two zinc fingers contribute synergistically to the binding to DNA, indicating that both fingers are necessary for the high affinity DNA binding.
EPF家族是一组转录因子，包含典型的Cys2/His2型锌指基序，最初在植物中发现。这些锌指蛋白的特征是两个锌指被不同长度的间隔区隔开，这些间隔区比簇状锌指蛋白中的典型间隔区（HC连接）长得多。我们在此描述了直接证据，表明这两个锌指与两个串联重复的AGT核心序列接触，这两个核心序列相隔约13个碱基对，这与结合相邻三联体序列的簇状锌指蛋白形成对比。DNA结合亲和力对核心序列之间的间隔敏感，并且对间隔的敏感性受到核心序列之间DNA序列的极大影响，富含GC的序列赋予的特异性比富含AT的序列高得多。在EPF家族成员中，EPF1对间隔的敏感性低于EPF2-5。这些结果表明，EPF不仅通过核心位点的序列识别其同源靶DNA，还通过核心位点之间的间隔识别，此外，EPF家族中的不同成员通过参考这两个参数来区分其特定的靶基因。这代表了Cys2/His2型锌指转录因子中一种独特的靶序列识别类型。此外，定点诱变研究表明，这两个锌指协同作用于与DNA的结合，表明两个锌指对于高亲和力DNA结合都是必需的。
