Hiratsuka K, Wu X, Fukuzawa H, Chua N H
Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021-6399.
Plant Cell. 1994 Dec;6(12):1805-13. doi: 10.1105/tpc.6.12.1805.
We isolated and characterized an Arabidopsis cDNA encoding the DNA binding protein GT-1. This protein factor, which contains 406 amino acids, is highly homologous to the previously described tobacco DNA binding protein GT-1a/B2F but is 26 amino acids longer. Recombinant Arabidopsis GT-1, which was obtained from in vitro translation, bound to probes consisting of four copies of pea small subunit of ribulose bisphosphate carboxylase rbcS-3A box II and required the same GGTTAA core binding site as the binding activity of an Arabidopsis nuclear protein preparation. However, unlike the truncated tobacco GT-1a prepared from Escherichia coli extracts, the full-length Arabidopsis GT-1 bound to pea rbcS-3A box III and Arabidopsis chlorophyll a/b binding protein CAB2 light-responsive elements, both of which contain GATA motifs. Deletion and mutational analyses suggested that the predicted trihelix region of GT-1 is essential for DNA binding. Moreover, GT-1 binds to target DNA as a dimer, and its C-terminal region contains a putative dimerization domain that enhances the binding activity. Transient expression of the GT-1::beta-glucuronidase fusion protein in onion cells revealed the presence of a nuclear localization signal(s) within the first 215 amino acids of GT-1.
我们分离并鉴定了一个编码DNA结合蛋白GT-1的拟南芥cDNA。该蛋白因子含有406个氨基酸，与先前描述的烟草DNA结合蛋白GT-1a/B2F高度同源，但长26个氨基酸。通过体外翻译获得的重组拟南芥GT-1与由四个拷贝的豌豆核酮糖二磷酸羧化酶小亚基rbcS-3A框II组成的探针结合，并且需要与拟南芥核蛋白制剂的结合活性相同的GGTTAA核心结合位点。然而，与从大肠杆菌提取物中制备的截短烟草GT-1a不同，全长拟南芥GT-1与豌豆rbcS-3A框III和拟南芥叶绿素a/b结合蛋白CAB2光响应元件结合，这两者都含有GATA基序。缺失和突变分析表明，GT-1预测的三螺旋区域对于DNA结合至关重要。此外，GT-1以二聚体形式结合靶DNA，其C末端区域含有一个增强结合活性的假定二聚化结构域。GT-1::β-葡萄糖醛酸酶融合蛋白在洋葱细胞中的瞬时表达揭示了GT-1前215个氨基酸内存在核定位信号。
