Pierrou S, Hellqvist M, Samuelsson L, Enerbäck S, Carlsson P
Department of Molecular Biology, Göteborg University, Sweden.
EMBO J. 1994 Oct 17;13(20):5002-12. doi: 10.1002/j.1460-2075.1994.tb06827.x.
The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.
叉头结构域是一种单体DNA结合基序，它定义了一个快速增长的真核转录调节因子家族。遗传和生化数据表明，编码叉头蛋白的基因在胚胎发育中起核心作用。我们利用聚合酶链反应（PCR）和低严谨度杂交技术，从人cDNA和基因组文库中分离出代表7个新的叉头基因（freac-1至freac-7）的克隆。这7个freac基因的表达空间模式从单一组织特异性到几乎普遍存在不等。通过从随机序列寡核苷酸中选择结合位点，确定了4种FREAC蛋白的DNA结合特异性。所有4种FREAC蛋白的结合位点都有一个核心序列RTAAAYA，但核心两侧的位置不同。两种FREAC蛋白之间的结构域交换确定了叉头结构域内的两个亚区域，它们造成了DNA结合特异性的差异。应用循环置换分析，我们发现FREAC蛋白与其同源位点的结合会导致DNA弯曲80 - 90度。
