Hope College, Department of Chemistry, 35 E. 12th Street, Holland, MI, 49423, United States.
Rutgers University, Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, 683 Hoes Lane, Piscataway, NJ, 08854, United States.
Biochem Biophys Res Commun. 2020 Jul 30;528(3):580-585. doi: 10.1016/j.bbrc.2020.05.141. Epub 2020 Jun 3.
Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial gene expression must be coordinated with nuclear gene expression to respond to cellular energetic needs. To gain insight into the coordination between the nucleus and mitochondria, there is a need to understand the regulation of transcription of mitochondrial DNA (mtDNA). Reversible protein post-translational modifications of the mtDNA transcriptional machinery may be one way to control mtDNA transcription. Here we focus on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, serine 197, and threonine 313. Phosphomimetics were established at these positions. Proteins were purified and analyzed for their ability to bind mtDNA and initiate transcription in vitro. Our results indicate phosphorylation at threonine 184 and threonine 313 impairs promoter binding and prevents transcription. These findings provide a potential regulatory mechanism of mtDNA transcription and help clarify the importance of protein post-translational modifications in mitochondrial function.
哺乳动物细胞的遗传信息存在于两个区域，细胞核和线粒体。线粒体基因的表达必须与核基因表达相协调，以响应细胞的能量需求。为了深入了解核与线粒体之间的协调，有必要了解线粒体 DNA（mtDNA）转录的调控。mtDNA 转录机制的可逆蛋白质翻译后修饰可能是控制 mtDNA 转录的一种方式。在这里，我们专注于 mtDNA 转录起始复合物的一个成员，线粒体转录因子 B2（TFB2M）。TFB2M 在启动子处使 mtDNA 解链，以便 RNA 聚合酶（POLRMT）能够访问 DNA 模板并起始转录。先前通过质谱法在 TFB2M 上鉴定了三个磷酸化位点：苏氨酸 184、丝氨酸 197 和苏氨酸 313。在这些位置建立了磷酸模拟物。纯化蛋白质并分析其结合 mtDNA 和体外起始转录的能力。我们的结果表明，磷酸化苏氨酸 184 和苏氨酸 313 会损害启动子结合并阻止转录。这些发现为 mtDNA 转录的潜在调节机制提供了依据，并有助于阐明蛋白质翻译后修饰在线粒体功能中的重要性。
