PLANT BZIP PROTEIN-DNA BINDING-SPECIFICITY
PLANT BZIP PROTEIN-DNA BINDING-SPECIFICITY
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DOI：
10.1006/jmbi.1993.1230
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发表时间：
1993-04-20
影响因子：
5.6
通讯作者：
CHUA, NH
中科院分区：
文献类型：
--
作者：
IZAWA, T;FOSTER, R;CHUA, NH
植物bZIP蛋白对含有ACGT核心的DNA序列基序表现出松弛的DNA结合特异性。凝胶迁移率变动实验，采用10种不同的重组植物bZIP蛋白表明，核苷酸侧翼的ACGT核心影响结合特异性，并确定了三种不同类型的ACGT元件：G盒，CACGTG; C盒，GACGTC;和A盒，TACGTA，基序。这10种不同的bZIP蛋白可以根据它们对G-box和C-box元件的定性和定量特异性分为三组。解离常数值（Kd值），这些bZIP蛋白的高亲和力G-盒和C-盒元件和互惠竞争凝胶迁移率变动测定证实了我们的分类方案。第1组蛋白对G盒元件表现出更强的结合亲和力，第2组蛋白以相当的结合亲和力结合G盒和C盒基序，而第3组蛋白对C盒寡核苷酸表现出更强的结合亲和力。使用一组侧翼序列不同的G盒和C盒寡核苷酸的研究鉴定了高亲和力结合位点。所有十个植物bZIP蛋白检查，除了TGA 1a，表现出A型G盒结合活性，首选I类G盒元件。与G盒元件观察到的情况相反，C盒基序显示出非常严格的侧翼核苷酸结合活性的要求。使用高亲和力G盒元件和G盒/C盒杂合元件的扫描突变体的蛋白/DNA结合实验表明，bZIP蛋白结合活性取决于蛋白二聚体亚基对ACGT半位点的亲和力。通过我们对植物bZIP DNA结合特异性的系统分析所提供的信息可以用于识别这里研究的植物bZIP蛋白的高亲和力结合位点。假设只有高亲和力的bZIP结合位点可能在体内起作用，这些位点的鉴定将使我们能够预测哪些基因被特定的bZIP蛋白激活。
Plant bZIP proteins exhibit a relaxed DNA-binding specificity for DNA sequence motifs containing an ACGT core. Gel mobility shift experiments employing ten different recombinant plant bZIP proteins demonstrated that nucleotides flanking the ACGT core affected binding specificity and identified three different types of ACGT elements: G-box, CACGTG; C-box, GACGTC; and A-box, TACGTA, motifs. These ten different bZIP proteins could be categorized into three groups according to their qualitative and quantitative specificity for G-box and C-box elements. Dissociation constant values (Kdvalues) of these bZIP proteins for high affinity G-box and C-box elements and reciprocal competition gel mobility shift assays confirmed our classification scheme. Group 1 proteins exhibit a stronger binding affinity for G-box elements, group 2 proteins bind both G-box and C-box motifs with comparable binding affinity, whereas the group 3 proteins display a stronger binding affinity for C-box oligonucleotides. Studies using a panel of G-box and C-box oligonucleotides differing in their flanking sequences identified high affinity binding sites. All ten plant bZIP proteins examined, except TGA1a, exhibited type A G-box binding activity preferring class I G-box elements. In contrast to the situation observed for G-box elements, C-box motifs displayed a very much more stringent flanking nucleotide requirement for binding activity. Protein/DNA binding experiments using scanning mutants of a high affinity G-box element and G-box/C-box hybrid elements demonstrated that bZIP protein binding activity depends upon the affinity of protein dimer subunits for ACGT half-sites. Information provided by our systematic analysis of plant bZIP DNA binding specificity can be used to identify high affinity binding sites for the plant bZIP proteins studied here. Assuming that only high affinity bZIP binding sites are likely to functionin vivo, identification of these sites will allow us to predict which genes are activated by a particular bZIP protein.