The human mitochondrial transcription termination factor (mTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions
The human mitochondrial transcription termination factor (mTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions
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DOI：
10.1093/emboj/16.5.1066
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发表时间：
1997-03-03
期刊：
影响因子：
11.4
通讯作者：
Attardi, G
中科院分区：
文献类型：
--
作者：
FernandezSilva, P;MartinezAzorin, F;Attardi, G
人类线粒体转录终止因子 (mTERF) cDNA 已在体外克隆和表达，并且该蛋白质的两种替代前体已被导入分离的线粒体并加工成成熟蛋白质。前体含有线粒体靶向序列，成熟的 mTERF（342 个残基）具有三个亮氨酸拉链（其中一个是二分的）和两个间隔较宽的基本结构域。体外合成的成熟蛋白对含有指导终止所需的十三聚体序列的双链寡核苷酸具有预期的特异性结合能力，并产生与天然蛋白产生的非常相似的 DNase I 足迹。然而，与后者相反，它在体外系统中缺乏转录终止促进活性，这表明需要另一种成分来使 mTERF 具有终止能力。通过带位移分析对重组蛋白及其诱变版本进行了详细的结构功能分析，表明基本结构域和三个亮氨酸拉链基序对于 DNA 结合都是必需的。此外，各种测试表明，重组和天然mTERF均作为单体与DNA结合，反对亮氨酸拉链的二聚化作用，并且与诱变实验的结果一起指出，需要分子内亮氨酸拉链相互作用来使两个基本结构域与mTERF靶DNA序列紧密对准。
The human mitochondrial transcription termination factor (mTERF) cDNA has been cloned and expressed in vitro, and two alternative precursors of the protein have been imported into isolated mitochondria and processed to the mature protein. The precursors contain a mitochondrial targeting sequence, and the mature mTERF (342 residues) exhibits three leucine zippers, of which one is bipartite, and two widely spaced basic domains. The in vitro synthesized mature protein has the expected specific binding capacity for a double-stranded oligonucleotide containing the tridecamer sequence required for directing termination, and produces a DNase I footprint very similar to that produced by the natural protein. However, in contrast to the latter, it lacks transcription termination-promoting activity in an in vitro system, pointing to another component(s) being required for making mTERF termination-competent. A detailed structure-function analysis of the recombinant protein and mutagenized versions of it by band shift assays has demonstrated that both basic domains and the three leucine zipper motifs are necessary for DNA binding. Furthermore, a variety of tests have shown that both the recombinant and the natural mTERF bind to DNA as a monomer, arguing against a dimerization role for the leucine zippers, and rather pointing, together with the results of mutagenesis experiments, to intramolecular leucine zipper interactions being required to bring the two basic domains in close register with the mTERF target DNA sequence.