A METAL-DEPENDENT DNA-BINDING PROTEIN INTERACTS WITH A CONSTITUTIVE ELEMENT OF A LIGHT-RESPONSIVE PROMOTER
A METAL-DEPENDENT DNA-BINDING PROTEIN INTERACTS WITH A CONSTITUTIVE ELEMENT OF A LIGHT-RESPONSIVE PROMOTER
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DOI：
10.1105/tpc.2.9.857
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发表时间：
1990-09-01
期刊：
影响因子：
11.6
通讯作者：
CHUA, NH
中科院分区：
文献类型：
--
作者：
LAM, E;KANOMURAKAMI, Y;CHUA, NH
我们使用DNase I足迹来表征与豌豆rb - s - 3a的光响应启动子结合的核因子，rb - s - 3a是编码核酮糖-1,5-二磷酸羧化酶小亚基的基因家族的一个成员。序列特异性结合活性，被称为3AF1，与rbcS-3A启动子-45区域的一个富含at的序列结合。3AF1结合位点的四聚体（Box VI）可与烟叶和根核提取物形成多个配合物。Box VI中3个碱基对的突变严重减少了体外dna -蛋白复合物的形成。当将野生型Box VI四聚体置于花椰菜花叶病毒35S启动子的上游时，该四聚体在转基因烟草中具有活性，而突变型四聚体则没有。这些结果将3AF1的结合与Box VI的体内功能联系起来。Box VI四聚体/35S嵌合结构在多种细胞类型和器官中表达，其活性不依赖于光。通过Box VI四聚体作为探针筛选cDNA表达文库，我们获得了一个推测的3AF1 dna结合活性cDNA克隆。表达该cDNA克隆的大肠杆菌溶原提取物可获得序列特异性复合物（Box VI）。由该cDNA编码的蛋白质的推导出的氨基酸序列包含约100个残基的两个片段，同源性为80%。此外，在每一个重复序列中，都有组氨酸和半胱氨酸的排列，这可能与许多dna结合蛋白中发现的锌指基序的两种已知类型有关。与金属配位在该蛋白结合DNA中起重要作用的预期一致，我们发现1,10-菲罗啉可以消除DNA-蛋白复合物的形成。有趣的是，我们发现同样的处理并没有消除烟草粗核提取物中3AF1的DNA结合活性。这些数据表明，核3AF1活性可能是由于多个dna结合蛋白在体外与Box VI相互作用所致。RNA凝胶印迹分析表明，与该cDNA同源的多个转录本在烟草不同器官中均有表达。
We have used DNase I footprinting to characterize nuclear factors that bind to the light-responsive promoter of pea rbc-S-3A, one member of the gene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. A sequence-specific binding activity, designated 3AF1, binds to an AT-rich sequence present at the -45 region of the rbcS-3A promoter. A tetramer of the 3AF1 binding site, designated as Box VI, can form multiple complexes with tobacco leaf and root nuclear extracts. Mutations of 3 base pairs in Box VI severely reduce DNA-protein complex formation in vitro. The wild-type Box VI tetramer, but not the mutant tetramer, is active in transgenic tobacco plants when placed upstream of the cauliflower mosaic virus 35S promoter truncated at -90. These results correlate binding of 3AF1 to the in vivo function of Box VI. The Box VI tetramer/35S chimeric construct confers expression in diverse cell types and organs and its activity is not dependent on light. By using the Box VI tetramer as a probe to screen a cDNA expression library, we have obtained a putative cDNA clone for the 3AF1 DNA-binding activity. Lysogen extracts of Escherichia coli expressing the cDNA clone give sequence-specific complexes with Box VI. The deduced amino acid sequence of the protein encoded by the cDNA contains two stretches of about 100 residues that are 80% homologous. Moreover, in each of the two repeats, there is an arrangement of histidines and cysteines, which may be related to the two known types of zinc-finger motifs found in many DNA-binding proteins. Consistent with the expectation that metal coordination plays an important role in DNA binding by this protein, we found that 1,10-phenanthroline can abolish the formation of DNA-protein complexes. Interestingly, we found that the same treatment did not abolish the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco. These data indicate that the nuclear 3AF1 activity is likely due to multiple DNA-binding proteins all interacting with Box VI in vitro. RNA gel blot analysis shows that multiple transcripts homologous to this cDNA are expressed in different tobacco organs.