Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5 S RNA gene.
Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5 S RNA gene.
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DOI：
10.1016/0022-2836(92)90248-i
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发表时间：
1992-02
影响因子：
5.6
通讯作者：
X. Liao;K. Clemens;L. Tennant;P. Wright;J. Gottesfeld
中科院分区：
文献类型：
--
作者：
X. Liao;K. Clemens;L. Tennant;P. Wright;J. Gottesfeld
通过聚合酶链反应扩增Xenopus 5 S RNA基因特异性转录因子IIIA（TFIIIA）cDNA的前101个氨基酸残基，获得编码该区域的DNA质粒。该多肽包括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大于95%的均一性。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。结合是金属依赖性的，用EDTA处理多肽消除结合。在相同条件下测得的多肽-DNA复合物的解离常数为5.6（± 0.9）nm，而全长XenopusTFIIIA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。该多肽保护内部控制区的21个碱基对免受DNA酶I的攻击，保护120个碱基对基因的+75至+95核苷酸。该区域包括C区启动子元件和几个对TFIIIA结合必不可少的鸟嘌呤残基。甲基化干扰实验表明，多肽和TFIIIA的结合模式是相似的。使用一系列合成的双链脱氧核糖寡核苷酸确定多肽结合所需的最小DNA序列。跨越5 S RNA基因+80至+92核苷酸的13个碱基对寡核苷酸保留了特异性和高亲和力结合，尽管后者相对于较长的DNA片段减少了6倍。含有TFIIIA的指1和2或指2、3和4的多肽不表现出序列特异性DNA结合。总的来说，这些研究为一个模型提供了强有力的支持，在该模型中，TFIIIA的前三个锌指以高亲和力结合在5 S RNA基因的内部控制区的核苷酸+80和+92之间。
A DNA plasmid encoding the first 101 amino acid residues of theXenopus5 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 inEscherichia coliand 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-lengthXenopusTFIIIA 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.