Guiltinan M J, Miller L
Pennsylvania State University, Department of Horticulture, University Park 16802.
Plant Mol Biol. 1994 Nov;26(4):1041-53. doi: 10.1007/BF00040687.
The wheat basic-leucine zipper (bZIP) DNA-binding protein EmBP-1 has been implicated in the mechanisms of abscisic acid (ABA) mediated gene regulation. Sequence and structural homology to the yeast bZIP protein GCN4 has been used to predict the location of the functional domains of EmBP-1. In order to test these predictions, the presumptive DNA-binding and dimerization domains of EmBP-1 were mapped by producing a series of truncated protein fragments and functionally testing them in vitro. Deletion of 5 amino acids of the predicted basic domain resulted in a loss of all DNA-binding activity. A fragment containing all six leucine repeat elements showed strong DNA-binding activity. Sequential deletion of the leucine repeat elements resulted in first an increase in DNA-binding activity (-L6 and -L5) followed by a reduction in binding activity (-L4) and eventually complete elimination of all detectable DNA-binding activity (-L3 and -L2). This demonstrates the importance of an intact leucine zipper domain of at least 4 repeat elements for efficient DNA-binding. The smallest polypeptide that retained DNA-binding activity is a fragment spanning amino acid residues 248-308 (ca. 8.4 kDa) consisting of minimal basic and leucine zipper domains. Dimerization of EmBP-1 was demonstrated by co-translation of fragments of differing molecular weights and identification of a DNA-protein complex with intermediate mobility to that produced by each fragment alone. A unique leucine-proline repeat element found N-terminal to the DNA-binding domain of EmBP-1 does not appear to play a role in DNA-binding or dimerization. These results confirm the locations of the functional domains of EmBP-1 predicted by similarity to GCN4. The high degree of functional conservation of the bZIP proteins spanning organisms from plants to fungi highlights the ancient origin of this class of transcription factors and of the mechanisms of gene regulation in which they participate.
小麦碱性亮氨酸拉链（bZIP）DNA结合蛋白EmBP-1与脱落酸（ABA）介导的基因调控机制有关。通过与酵母bZIP蛋白GCN4的序列和结构同源性来预测EmBP-1功能域的位置。为了验证这些预测，通过产生一系列截短的蛋白片段并在体外对其进行功能测试，绘制了EmBP-1假定的DNA结合和二聚化结构域。预测的碱性结构域缺失5个氨基酸会导致所有DNA结合活性丧失。包含所有六个亮氨酸重复元件的片段显示出很强的DNA结合活性。亮氨酸重复元件的顺序缺失首先导致DNA结合活性增加（-L6和-L5），随后结合活性降低（-L4），最终完全消除所有可检测到的DNA结合活性（-L3和-L2）。这证明了至少4个重复元件的完整亮氨酸拉链结构域对于有效DNA结合的重要性。保留DNA结合活性的最小多肽是一个跨越氨基酸残基248 - 308（约8.4 kDa）的片段，由最小的碱性和亮氨酸拉链结构域组成。通过共翻译不同分子量的片段并鉴定出一种迁移率介于每个片段单独产生的DNA - 蛋白质复合物之间的DNA - 蛋白质复合物，证明了EmBP-1的二聚化。在EmBP-1的DNA结合结构域N端发现的一个独特的亮氨酸 - 脯氨酸重复元件似乎在DNA结合或二聚化中不起作用。这些结果证实了通过与GCN4相似性预测的EmBP-1功能域的位置。从植物到真菌的生物体中bZIP蛋白的高度功能保守性突出了这类转录因子及其参与的基因调控机制的古老起源。
