Regulation of Mitochondrial DNA

*线粒体在细胞功能中发挥的作用越来越被研究人员认识到。线粒体编码它们自己的基因组(MtDNA)，这是一个对它们的功能至关重要的分子。尽管首次描述线粒体DNA是在50多年前，但关于线粒体DNA是如何维持和表达的，仍然存在许多问题。作为一名研究线粒体调控基本机制的研究人员，我新设立的项目将探索线粒体DNA的调控。除了传统的研究线粒体和线粒体DNA的方法外，我们还将利用我们开发的新方法将DNA导入线粒体。利用我们在线粒体DNA和线粒体细胞生物学方面的专业知识，拟议的研究包括三个目标，总体目标是了解蛋白质和序列元件如何调节线粒体DNA。*目标1：确定线粒体DNA的平衡表达如何控制*三个线粒体启动子的相对转录速率平衡线粒体基因表达、线粒体DNA复制和线粒体核糖体生物发生。我们将通过研究线粒体转录因子和mtDNA序列的作用来确定线粒体转录的相对速度是如何调节的。此外，我们将研究影响线粒体转录的信号通路如何改变线粒体表达的平衡，并确定改变这种平衡的功能后果。*目标2：测试mtDNA序列元件在细胞中的作用*我们将DNA导入线粒体的新方法使我们能够首次测试mtDNA序列元件的细胞相关性。我们将不再直接操纵mtDNA，而是将含有mtDNA调控元件的质粒转化为可以工程设计的。确定维持该质粒所需的结构元件将导致对mtDNA如何调控的新的理解。*目标3：揭示核转录因子在线粒体中的新作用*虽然已有核转录因子(NTF)在线粒体中的报道，但它们的作用在很大程度上仍未确定。神经营养因子是否在线粒体转录中起直接作用还存在争议，因为很难将它们的核功能和线粒体功能分开。我们将通过将这些转录因子直接定位于线粒体并确定它们对线粒体功能的影响来测试这个问题。*新颖性和意义*我们的研究是我们以前工作的延伸，将通过回答有关线粒体DNA调控的基本问题来促进我们对线粒体功能的基本理解。提高我们对线粒体的基本了解将在细胞和生物层面产生影响，也与动物健康有关。这项研究计划还将为下一波线粒体研究人员提供培训场所。********

***The role that mitochondria play in cellular function is increasingly being recognized by researchers. Mitochondria encode their own genome (mtDNA), a molecule that is essential for their function. Although first described over 50 years ago, many questions remain about how mtDNA is maintained and expressed. As a researcher studying the basic mechanisms regulating mitochondria, my newly established program will explore mtDNA regulation. In addition to traditional means of studying mitochondria and mtDNA, we will utilize novel methods we have developed to transfect DNA into mitochondria. Taking advantage of our expertise in mtDNA and mitochondrial cell biology, the proposed research comprises three aims with an overall goal of understanding how proteins and sequence elements regulate mtDNA. ******Aim 1: To determine how the balanced expression of mtDNA is controlled ***The relative rates of transcription from the three mitochondrial promoters balances mitochondrial gene expression, mtDNA replication, and mitochondrial ribosome biogenesis. We will determine how the relative rates of mitochondrial transcription are regulated by examining the roles of mitochondrial transcription factors and mtDNA sequences. Additionally, we will examine how signaling pathways affecting mitochondrial transcription alter the balance of mitochondrial expression and also determine the functional consequences of altering this balance. ******Aim 2: To test the role of mtDNA sequence elements in cells ***Our novel methods for transfecting DNA into mitochondria allow us for the first time to test the cellular relevance of mtDNA sequence elements. Instead of directly manipulating mtDNA, which is currently not possible, we will transfect a plasmid containing mtDNA regulatory elements, which can be engineered. Determining which structural elements are required for the maintenance of this plasmid will lead to novel understanding about how the mtDNA is regulated. ******Aim 3: To uncover novel roles of nuclear transcription factors in mitochondria ***While nuclear transcription factors (NTFs) have been reported in mitochondria, their roles remain largely undefined. Whether NTFs play a direct role in mitochondrial transcription is a matter of debate, as it is difficult to separate their nuclear and mitochondrial functions. We will test this issue by targeting these transcription factors directly to mitochondria and determining their effects on mitochondrial function. ******Novelty and Significance ***Our studies, which are an extension of our previously work, will advance our basic understanding of mitochondrial function by answering fundamental questions about the regulation of mtDNA. Improving our basic understanding of mitochondria will have an impact at both the cellular and organismal level and is also relevant to animal health. This research program will also provide a training venue for the next wave of mitochondrial researchers. ********