The mammalian citochondrial code

描述(由申请人提供)：这项工作旨在了解哺乳动物线粒体中甲硫氨酸(Met)和异亮氨酸(Ile)的不寻常遗传密码是如何表达的。导致这种表达的线粒体tRNAMet和tRNAIle突变与Leber遗传性视神经病变以及许多线粒体肌肉疾病和神经肌肉疾病有关。线粒体密码的一个独特特征是将AUA密码子分配给Met而不是Ile。除了标准的Aug密码子之外，Met的第二个密码子的增加似乎是一种适应机制，以增加蛋白质中Met的含量，以响应哺乳动物线粒体的高度氧化环境。这是通过在线粒体tRNAMet的CAU反密码子的摆动位置对C34(F5C34)进行新的5-甲酰化修饰，以及通过线粒体tRNAIle的GAU反密码子缺乏修饰而实现的，该反密码子只读取ILE的AUU和AUC。我们将综合运用酶学、生物信息学、蛋白质纯化和动力学分析等多学科的方法来阐明确保Met和Ile在哺乳动物线粒体中准确表达的分子机制。在目标1中，对潜在的5-甲酰基的一碳供体进行了化学合成和生化分析相结合的方法来确定f5C34的生物合成途径。将结合蛋白质纯化、比较基因组学和候选基因产物的生化分析来鉴定参与该途径的酶。这将为研究这些新的酶的机制和建立生物信息学模型来确定它们在线粒体tRNAMet相关疾病中的可能作用开辟了道路。在目标2中，实验将解决线粒体tRNAMet和tRNAIle中反密码子修饰的状态如何决定偏差密码的准确性。由线粒体Met-和Ile-tRNA合成酶催化的tRNA氨基酰化的准确性和由线粒体核糖体催化的tRNA解码的准确性将被用酶学和动力学的综合方法来研究。将开发新的分析方法来重建线粒体核糖体并研究解码步骤，从而建立一个类似于细菌核糖体研究中使用的体外翻译系统。这些实验的目的是为了定量了解线粒体基因表达的准确性。这项工作的意义很大，因为它从根本上解决了为什么线粒体tRNA的突变和缺乏修饰与100多种线粒体疾病有关。 公共卫生相关性：哺乳动物线粒体遗传密码对公共健康的重要性是基于发现了100多种与线粒体疾病相关的人类遗传病。这些疾病中的大多数与线粒体tRNAs的突变或其核苷酸修饰的缺陷有关。这项工作旨在了解线粒体密码表达中导致人类线粒体tRNAMet和tRNAIle异常解码的分子机制，与线粒体肌病、脑肌病、高血压和肥厚性心肌病以及Leber遗传性视神经病变直接相关。

DESCRIPTION (provided by applicant): This work seeks to understand how the unusual genetic code for methionine (Met) and isoleucine (Ile) in mammalian mitochondria is expressed. Mutations in mitochondrial tRNAMet and tRNAIle responsible for the expression are linked to Leber's hereditary optic neuropathy and numerous mitochondrial myopathies and neuromuscular disorders. A unique feature of the mitochondrial code is the assignment of the AUA codon to Met instead of Ile. This addition of a second codon for Met, in addition to the standard AUG codon, appears to be an adaptive mechanism to increase Met contents in proteins in response to the highly oxidative environment of mammalian mitochondria. It is accomplished by the presence of a novel 5-formyl modification to C34 (f5C34) in the wobble position of the CAU anticodon of mitochondrial tRNAMet and by the lack of modification in the GAU anticodon of mitochondrial tRNAIle, which reads only AUU and AUC for Ile. A multidisciplinary approach, consisting of enzymology, bioinformatics, protein purification, and kinetic analysis, will be used to elucidate the molecular mechanisms that ensure accurate expression of Met and Ile in mammalian mitochondria. In aim 1, a combination of chemical synthesis and biochemical analysis of potential one-carbon donors of the 5-formyl group is undertaken to determine the biosynthetic pathway to f5C34. Enzymes involved in the pathway will be identified using a combination of protein purification, comparative genomics, and biochemical analysis of candidate gene products. This will open the way to study the mechanisms of these novel enzymes and to build bioinformatics models for determining their possible roles in mitochondrial tRNAMet-related diseases. In aim 2, experiments will address how the status of anticodon modification in mitochondrial tRNAMet and tRNAIle determines the accuracy of the deviant code. Both the accuracy of tRNA aminoacylation, catalyzed by the mitochondrial Met- and Ile-tRNA synthetases, and the accuracy of tRNA decoding, catalyzed by the mitochondrial ribosome, will be interrogated in a comprehensive enzymatic and kinetic approach. New assays will be developed to reconstitute the mitochondrial ribosome and to investigate the decoding step, thus establishing an in vitro translation system similar to the one used in studies of the bacterial ribosome. These experiments are designed with a view towards a quantitative understanding of the accuracy of mitochondrial gene expression. The significance of the work is high in that it addresses at the fundamental level why mutations and lack of modification in mitochondrial tRNAs are associated with over 100 mitochondrial disorders. PUBLIC HEALTH RELEVANCE: The importance of the mammalian mitochondrial genetic code to public health is based on the discovery of over 100 human genetic diseases associated with mitochondrial disorders. The majority of these diseases are linked to mutations in mitochondrial tRNAs or to deficiencies in their nucleotide modifications. This work seeks to understand the molecular mechanisms responsible for the unusual decoding of human mitochondrial tRNAMet and tRNAIle in the expression of the mitochondrial code, with direct relevance to mitochondrial myopathies, encephalomyopathy, hypertension and hypertrophic cardiomopathy, and Leber's hereditary optic neuropathy.