Department of Molecular Pathogenesis, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Methods Mol Biol. 2023;2615:139-151. doi: 10.1007/978-1-0716-2922-2_11.
Mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA)-binding protein that plays a crucial dual role in the initiation of mitochondrial transcription initiation and mtDNA maintenance. Because TFAM directly interacts with mtDNA, assessing its DNA-binding property can provide useful information. This chapter describes two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay with recombinant TFAM proteins, which both require simple agarose gel electrophoresis. These are used to investigate the effects of mutations, truncation, and posttranslational modifications on this key mtDNA regulatory protein.
线粒体转录因子 A（TFAM）是一种与线粒体 DNA（mtDNA）结合的蛋白质，它在启动线粒体转录起始和 mtDNA 维持方面发挥着至关重要的双重作用。由于 TFAM 直接与 mtDNA 相互作用，评估其 DNA 结合特性可以提供有用的信息。本章描述了两种体外测定方法，即电泳迁移率变动分析（EMSA）和使用重组 TFAM 蛋白的 DNA 解链分析，这两种方法都需要简单的琼脂糖凝胶电泳。这些方法可用于研究突变、截短和翻译后修饰对这种关键的 mtDNA 调节蛋白的影响。
