Zeller R W, Coffman J A, Harrington M G, Britten R J, Davidson E H
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Dev Biol. 1995 Jun;169(2):713-27. doi: 10.1006/dbio.1995.1181.
Several Strongylocentrotus purpuratus gene cis-regulatory regions contain asymmetric C4 sequences which are core elements of target sites for a specific DNA-protein interaction. Blastula stage nuclear extract contains five proteins which specifically bind to these target sites, resulting in a characteristic pattern of complexes in gel mobility shift assays. We used automated affinity chromatography to purify a protein which binds to these sites and have isolated the corresponding cDNA. This protein, SpGCF1, is a novel sea urchin DNA-binding protein with no overall homology to proteins reported in the databases currently available. The DNA-binding domain of this protein was identified by a deletion analysis. As demonstrated both for protein translated in vitro and for bacterial protein expressed from a cDNA clone, a single SpGCF1 mRNA serves as a template for the synthesis of five DNA-binding polypeptides. We show that these five polypeptides are most likely produced by differential usage of a nested set of AUG start codons in the SpGCF1 cDNA and thus contain variable amounts of a proline-rich N-terminal domain. Since proline-rich regions often serve as transcriptional activation domains, the five SpGCF1 proteins apparently possess different "activation potentials."
几种紫球海胆基因顺式调控区域含有不对称的C4序列，这些序列是特定DNA - 蛋白质相互作用靶位点的核心元件。囊胚期核提取物含有五种能特异性结合这些靶位点的蛋白质，在凝胶迁移率变动分析中产生特征性的复合物模式。我们使用自动亲和层析法纯化了一种能结合这些位点的蛋白质，并分离出了相应的cDNA。这种蛋白质SpGCF1是一种新型的海胆DNA结合蛋白，与目前可用数据库中报道的蛋白质没有整体同源性。该蛋白质的DNA结合结构域通过缺失分析得以鉴定。正如体外翻译的蛋白质和从cDNA克隆表达的细菌蛋白所显示的那样，单个SpGCF1 mRNA作为合成五种DNA结合多肽的模板。我们表明，这五种多肽很可能是由SpGCF1 cDNA中一组嵌套的AUG起始密码子的差异使用产生的，因此含有不同数量的富含脯氨酸的N端结构域。由于富含脯氨酸的区域通常作为转录激活结构域，这五种SpGCF1蛋白显然具有不同的“激活潜力”。
