Studies on Transfer RNA

描述(申请人提供)：氨基酰-tRNAs提供了基因DNA序列中编码的遗传信息和相应蛋白质的氨基酸序列之间的接口。已发现绝大多数生物体至少缺少一种典型的氨基酰-tRNA合成酶。相应的氨基酰基-tRNA由非规范的氨基酰基-tRNA合成酶或通过另一种途径合成。这项拟议工作的一个广泛目标是从结构、生物化学和遗传角度表征参与这些新的氨酰-tRNA合成途径的酶和多酶及tRNA复合体，然后可用于揭示人类病原体的抗感染靶点和非天然氨基酸的掺入。这些总体目标将在拟议工作的四个具体领域实现。(I)Selenocyste-inyl-tRNA的形成是人类的一个基本过程，可能在某些病原体中也是如此。对所涉及的酶的研究，以及另一种尚不清楚的SEC-tRNA形成途径的研究，对于理解硒半胱氨酸的解码以及利用这一途径作为对抗昏睡病和疟疾的人类病原体具有重要意义。(Ii)利用基于结构的高通量随机突变和吡咯烷基-tRNA合成酶和tRNAPyl的计算设计，将构建几种赖氨酸衍生物的遗传编码系统，这对于揭示某些翻译后修饰在人类和其他真核生物中的作用将是重要的。此外，还将研究延伸因子EF-Tu对翻译质量的控制，同时还将努力设计能够特异性识别所需的非天然氨基酰tRNA的延伸因子突变体。(Iii)几种氨基酸(硒半胱氨酸、半胱氨酸、谷氨酰胺、天冬氨酸)在与tRNA结合的同时被合成。将研究氨酰-tRNA合成酶、tRNA和生物合成酶(S)之间的复合体的形成-可能对底物通道和保护翻译系统免受错误酰化的tRNA的影响-以揭示复合体的形成如何影响组成分子的酶行为。(Iv)我们对一种新的tRNA连接酶(S)的发现和拟议的表征将使我们更好地了解tRNA在人类和古生物中的加工过程。 与公共健康相关：氨酰-tRNA合成(维持DNA和蛋白质之间编码关系的过程)出人意料的多样性，在翻译和翻译后蛋白质修饰的生物学中开辟了以前无法触及的前沿，其故障与包括癌症、神经退行性疾病和代谢性疾病在内的几种人类疾病有关。这些拟议的项目旨在表征氨酰-tRNA形成的新途径和参与氨酰-tRNA合成的大分子复合体，以揭示人类病原体(包括昏睡病和疟疾的病原体)的抗感染靶点，并设计非天然氨基酸掺入的途径，用于理解翻译后蛋白质修饰在高等真核生物中的复杂作用。

DESCRIPTION (provided by applicant): Aminoacyl-tRNAs provide the interface between genetic information encoded in the DNA sequence of a gene and the amino acid sequence of the corresponding protein. The vast majority of organisms have been found to lack at least one canonical aminoacyl-tRNA synthetase. The corresponding amino acyl-tRNA is instead synthesized by either a non-canonical aminoacyl-tRNA synthetase or by an alternative pathway. A broad goal of the proposed work is to characterize structurally, biochemically and genetically enzymes and multi-enzyme and tRNA complexes involved in these new pathways for aminoacyl-tRNA synthesis that can then be used to uncover anti-infective targets for human pathogens and for unnatural amino acid incorporation. These general goals will be realized in four specific areas of the proposed work. (i) Selenocyste- inyl-tRNA formation is an essential process in humans, and likely in certain pathogens. Investigations of the enzymes involved, and of an alternative and still unknown route for Sec-tRNA formation will be of fundamental importance for understanding selenocysteine decoding and for exploiting this pathway as a way to combat human pathogens responsible for sleeping sickness and malaria. (ii) Using structure-based high-throughput random mutagenesis and computational design of pyrrolysyl-tRNA synthetase and tRNAPyl, genetic coding systems will be constructed for several lysine derivatives, which will be important for revealing the role of certain post-translational modifications in human and other eukaryotes. Translational quality control by the elongation factor EF-Tu will also be studied along side efforts to design elongation factor mutants that will specifically recognize the desired unnatural aminoacyl-tRNA. (iii) Several amino acids (selenocysteine, cysteine, glutamine, asparagine) are synthesized while attached to the tRNA. Complex formation, between the aminoacyl-tRNA synthetase, tRNA and biosynthetic enzyme(s)-possibly important for substrate channeling and protection of the translation system from mis-acylated tRNAs-will be investigated, to reveal how complex formation affects the enzymatic behavior of the component molecules. (iv) Our discovery and proposed characterization of a novel tRNA ligase(s) will lead to a better understanding of tRNA processing in humans and archaea. PUBLIC HEALTH RELEVANCE: The unexpected diversity of aminoacyl-tRNA synthesis (processes that maintain the coding relation- ship between DNA and protein) opens previously inaccessible frontiers in the biology of translation and post-translational protein modifications, the malfunction of which is linked to several human diseases including cancer, neurodegenerative and metabolic disorders. The proposed projects aim to characterize new routes for aminoacyl-tRNA formation and macromolecular complexes involved in aminoacyl-tRNA synthesis in order to uncover anti-infective targets for human pathogens (including the causative agents of sleeping sickness and malaria) and to design pathways for unnatural amino acid incorporation that will be applied to understanding the complex role of post-translational protein modifications in higher eukaryotes.