tRNase Z reaction is central to tRNA maturation

转移RNA(TRNA)是蛋白质合成的中心。与其他RNA一样，tRNA被转录为 前驱体，必须经过成熟。RNaseP去除5‘前导。核酸内切酶 TRNase Z去掉了3‘末端，这样CCA就可以被tRNA核苷酸转移酶加进去。 因此，tRNase Z反应是前tRNA成熟的关键步骤。挤压柔性臂(FA) 从远离活性部位的tRNaseZ的正文。球状FA手主要结合于 TRNA的肘部(D/T环)，远离剪切键。自然发生的突变 人类线粒体tRNAs与母体传播疾病和 症状，主要是肌病。[目的1]发病相关疾病的分布情况 线粒体tRNA突变与tRNA结构变化的相关性及其对血管紧张素转换酶的影响 TRNase Z处理？与发病机制相关的T环替换的影响将被调查。 [目的2]蛋白酶敏感性是否报告了tRNase Z FA的柔韧性？弥撒 光谱分析将揭示一种新的底物识别机制。 在此输入文本，它是您的应用程序的新的特定目标信息。推荐一页。 TRNA作为前体转录，并通过去除5‘前导和3’尾巴进行处理。 核糖核酸酶P内切法去除5‘前导。TRNase Z，前tRNA 3‘加工 内切酶，裂解3‘末端，在鉴别子上留下一个OH，即未配对的核苷酸 在受体茎的3‘侧，准备用于CCA加成。TRNase Z是？- 内酰胺酶家族金属依赖的水解酶。特异性识别tRNA的能力是 由柔性臂(FA)唯一授予tRNaseZ的球状手从和 通过一个结构化的茎连接到酶的身体上，它与T- 底物tRNA环(Li等人，2005,2006)。 目的1.致病相关线粒体tRNA突变的分布 与tRNA结构变化相关，以及对tRNase Z加工的影响？分析 线粒体T环致病相关突变对tRNase Z反应的影响 TRNA将与tRNA结构探测相结合。22个人类线粒体中的11个 编码的tRNA含有13个与致病相关的T环替换(参见MITOMAP：A Human 线粒体基因组数据库；http://www.mitomap.org，，2011年)。调查…的模式 对结构、加工和由此产生的病理的影响，我们将构建这些野生型 和突变型线粒体tRNAs及其取代对其结构的影响 和tRNase Z加工动力学。 目的2.蛋白酶敏感性是否报告了tRNase Z FA的柔韧性？删除 研究证实了FA在前tRNA底物识别中的功能(Schilling等人，2005年)。 FA对底物结合和特定FA残留量有两个数量级的贡献 和区域对于结合是重要的(Levinger等人，2009年)。为了调查足协的灵活性， 我们将使用蛋白酶敏感性和质谱学来比较野生型tRNaseZ和 几个重要的变异体，既在溶液中游离，又与前tRNA复合。

Transfer RNA (tRNA) is central to protein synthesis. Like other RNAs, tRNA is transcribed as a precursor and must undergo maturation. RNase P removes the 5' leader. The endonuclease tRNase Z removes the 3' trailer so that CCA can be added by tRNA nucleotidyltransferase. tRNase Z reaction is thus a critical step in pre-tRNA maturation. A flexible arm (FA) is extruded from the body of tRNase Z remote from the active site. The globular FA hand principally binds to the elbow (D/T loops) of tRNA, far from the scissile bond. Naturally occurring mutations in human mitochondrial tRNAs are associated with maternally transmitted diseases and syndromes, principally myopathies. [Aim 1] Does the distribution of pathogenesis-related mitochondrial tRNA mutations correlate with tRNA structure changes and effects on tRNase Z processing? Effects of pathogenesis-related T loop substitutions will be investigated. [Aim 2] Does protease susceptibility report on flexibility of the tRNase Z FA? The mass spectroscopic analysis will shed light on a novel mechanism of substrate recognition. Enter the text here that is the new specific aims information for your application. One page is recommended. tRNAs are transcribed as precursors and processed by removal of a 5' leader and a 3' trailer. RNase P endonucleolytically removes the 5' leader. tRNase Z, the pre-tRNA 3' processing endonuclease, cleaves the 3' trailer leaving an OH on the discriminator, the unpaired nucleotide at the 3' side of the acceptor stem, prepared for CCA addition. tRNase Z is a member of the ¿- lactamase family of metal-dependent hydrolases. Ability to specifically recognize tRNA is uniquely conferred upon tRNase Z by the flexible arm (FA), a globular hand extruded from and connected to the body of the enzyme by a structured stalk, which contacts nucleotides in the T- loop of substrate tRNA (Li et al., 2005, 2006). Aim 1. Does the distribution of pathogenesis-related mitochondrial tRNA mutations correlate with tRNA structure changes and effects on tRNase Z processing? Analysis of effects on tRNase Z reaction of pathogenesis-related mutations in the T-loops of mitochondrial tRNAs will be combined with tRNA structure probing. 11 of the 22 human mitochondrially encoded tRNAs harbor 13 pathogenesis-related T-loop substitutions (see MITOMAP: A Human Mitochondrial Genome Database; http://www.mitomap.org, 2011). To investigate patterns of effects on structure, processing and the resulting pathologies, we will construct these wild type and mutant mitochondrial tRNAs and characterize effects of the substitutions on their structure and tRNase Z processing kinetics. Aim 2. Does protease susceptibility report on flexibility of the tRNase Z FA? A deletion study demonstrated function of the FA in pre-tRNA substrate recognition (Schilling et al., 2005). The FA contributes two orders of magnitude toward substrate binding and specific FA residues and regions are important for binding (Levinger et al., 2009). To investigate flexibility of the FA, we will use protease susceptibility and mass spectrometry to compare wild type tRNase Z with several important variants, both free in solution and complexed with pre-tRNA.