Han K, Manley J L
Department of Biological Sciences, Columbia University, New York, New York 10027.
Genes Dev. 1993 Mar;7(3):491-503. doi: 10.1101/gad.7.3.491.
We have used a transient expression assay employing Drosophila tissue culture cells to study the transcriptional repression activity of the homeo domain protein Even-skipped (Eve). Eve was found to repress all promoters that contained Eve-binding sites, including both TATA-containing and TATA-lacking minimal promoters, as well as promoters activated by several different classes of activator proteins. These findings suggest that the general transcription machinery can be a target of Eve. By analyzing properties of a variety of Eve mutants and chimeric fusion proteins, we have identified several features important for efficient repression. In addition to the DNA-binding domain, a potent repressor requires a repression domain, which can be as small as 27 residues. The minimal 57-residue Eve repression domain, as well as several others studied here, were all found to be proline rich and to contain a high percentage of hydrophobic residues. An intriguing feature of the strong repressors was that their DNA-binding activities, measured by gel retention assays with nuclear extracts, were significantly less than those of derivatives inactive in repression.
我们利用果蝇组织培养细胞的瞬时表达试验来研究同源结构域蛋白偶数跳（Eve）的转录抑制活性。发现Eve能抑制所有包含Eve结合位点的启动子，包括含TATA盒和不含TATA盒的最小启动子，以及由几类不同激活蛋白激活的启动子。这些发现表明通用转录机制可能是Eve的作用靶点。通过分析多种Eve突变体和嵌合融合蛋白的特性，我们确定了几个对有效抑制很重要的特征。除了DNA结合结构域，一个有效的阻遏物还需要一个抑制结构域，其小至27个氨基酸残基。最小的57个氨基酸残基的Eve抑制结构域以及这里研究的其他几个结构域，都富含脯氨酸且含有高比例的疏水残基。强阻遏物的一个有趣特征是，用核提取物进行凝胶滞留试验测量时，它们的DNA结合活性明显低于在抑制方面无活性的衍生物。
