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TFIIIA的前三个锌指与非洲爪蟾5S RNA基因内部控制区的特异性相互作用。

Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5 S RNA gene.

作者信息

Liao X B, Clemens K R, Tennant L, Wright P E, Gottesfeld J M

机构信息

Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.

出版信息

J Mol Biol. 1992 Feb 20;223(4):857-71. doi: 10.1016/0022-2836(92)90248-i.

Abstract

A DNA plasmid encoding the first 101 amino acid residues of the Xenopus 5 S RNA gene-specific transcription factor IIIA (TFIIIA) was derived by polymerase chain reaction amplification of this region from the cDNA for TFIIIA. This polypeptide includes the first three zinc fingers of the TFIIIA DNA binding domain. The polypeptide was expressed in Escherichia coli and purified to greater than 95% homogeneity. The three finger polypeptide binds the internal control region of the 5 S RNA gene with sequence specificity and high affinity. Binding is metal-dependent and treatment of the polypeptide with EDTA abolishes binding. Polypeptide-DNA complexes exhibit a dissociation constant of 5.6(+/- 0.9) nM, while that for full-length Xenopus TFIIIA is 2.2(+/- 0.4) nM, measured under the same conditions. This suggests that the majority of the free energy of TFIIIA binding resides in these amino-terminal zinc fingers. The polypeptide protects 21 base-pairs of the internal control region from attack by DNase I, with protection from nucleotides +75 to +95 of the 120 base-pair gene. This region includes the C-block promoter element and several guanine residues that are essential for TFIIIA binding. Methylation interference experiments suggest that the mode of binding of the polypeptide and TFIIIA are similar. The minimal DNA sequences required for polypeptide binding were determined using a series of synthetic double-stranded deoxyribo-oligonucleotides. A 13 base-pair oligonucleotide spanning nucleotides +80 to +92 of the 5 S RNA gene retained specific and high-affinity binding, although the latter was reduced sixfold relative to longer DNA fragments. Polypeptides containing fingers 1 and 2, or fingers 2, 3 and 4 of TFIIIA do not exhibit sequence-specific DNA binding. Overall, these studies provide strong support for a model in which the first three zinc fingers of TFIIIA bind with high affinity between nucleotides +80 and +92 of the internal control region of the 5 S RNA gene.

摘要

通过聚合酶链反应从非洲爪蟾TFIIIA的cDNA中扩增该区域,获得了编码非洲爪蟾5S RNA基因特异性转录因子IIIA(TFIIIA)前101个氨基酸残基的DNA质粒。该多肽包含TFIIIA DNA结合结构域的前三个锌指。该多肽在大肠杆菌中表达并纯化至纯度大于95%。三指多肽以序列特异性和高亲和力结合5S RNA基因的内部控制区域。结合依赖于金属,用EDTA处理该多肽会消除结合。在相同条件下测量,多肽-DNA复合物的解离常数为5.6(±0.9)nM,而全长非洲爪蟾TFIIIA的解离常数为2.2(±0.4)nM。这表明TFIIIA结合的大部分自由能存在于这些氨基末端锌指中。该多肽保护内部控制区域的21个碱基对免受DNase I的攻击,保护范围为120个碱基对基因的核苷酸+75至+95。该区域包括C区启动子元件和几个对TFIIIA结合至关重要的鸟嘌呤残基。甲基化干扰实验表明该多肽和TFIIIA的结合模式相似。使用一系列合成的双链脱氧核糖寡核苷酸确定了多肽结合所需的最小DNA序列。一个跨越5S RNA基因核苷酸+80至+92的13个碱基对的寡核苷酸保留了特异性和高亲和力结合,尽管相对于更长的DNA片段,后者降低了六倍。包含TFIIIA的第1和第2个锌指或第2、3和4个锌指的多肽不表现出序列特异性DNA结合。总体而言,这些研究为TFIIIA的前三个锌指在5S RNA基因内部控制区域的核苷酸+80和+92之间高亲和力结合的模型提供了有力支持。

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