RNA modification: Structure and Mechanism

描述(由申请人提供)：转移RNA(TRNA)是翻译的关键分子，它们准确有效地解码遗传信息的能力取决于关键反密码子茎环(ASL)中核苷酸的转录后修饰。这些修饰的缺陷会导致遗传性人类线粒体疾病，而修饰的核苷可以作为敏感的诊断手段，例如人类癌症标记物。该研究项目的长期目标是详细了解ASL修饰的生物合成及其在细胞生理学中的作用，并确定这些途径中的新靶点以进行治疗干预。本申请的具体目的是从生化和结构上阐明通用修饰核苷苏氨酰氨基甲酰腺苷(T6A37)生物合成的分子机制。T6A37是一种复杂的、古老的ASL修饰，存在于所有tRNA中，对所有生命形式中的ANN密码子进行解码。它通过阻止核糖体框架移动，促进同源密码子识别、tRNA转位和合成酶识别，对tRNA的功能起着至关重要的作用。我们最近发现，TSAC、TSAB、TSAD和TSAE是细菌生物合成t6A37所必需的，也是足够的。由于这四种酶是细菌所必需的，其中两种是细菌领域特有的，t6A37途径是开发新一代抗菌疗法的一个引人注目的潜在靶点。在拟议的工作中，将通过包括动力学、生化、核磁共振和结晶学方法在内的多管齐下的方法来阐明该途径中第一种也是通用的酶TsaC的催化机制。同时，其他三种蛋白质在生物合成过程中的确切作用以及它们彼此之间以及它们与底物的协同作用将通过组合的方法来阐明，包括瞬时动力学实验、tRNA结合实验、核磁共振研究和X射线结晶学。这是一个多机构合作的项目，召集了跨学科的专业知识和方法来阐明T6A37生物合成的机制和结构基础，并为T6A37途径作为抗菌靶点的发展奠定了实验基础。

DESCRIPTION (provided by applicant): Transfer-RNAs (tRNA) are pivotal molecules of translation and their ability to accurately and efficiently decode genetic information depends on post-transcriptional modification of nucleotides in the critical anticodon stem loop (ASL). Deficiencies in these modifications cause hereditary human mitochondrial disease and modified nucleosides serve as sensitive diagnostics, such as human cancer markers. The long-term goal of this research project is to develop a detailed understanding of the biosynthesis of ASL modifications and their roles in cellular physiology, and to identify novel targets in these pathways for therapeutic intervention. This application specifically aims at elucidating biochemically and structurally the molecular mechanisms underlying the biosynthesis of the universal modified nucleoside threonylcarbamoyladenosine (t6A37). t6A37 is a complex, ancient modification of the ASL found in all tRNAs decoding ANN codons in all life forms. It is critical fo tRNA function by preventing ribosomal frame shifting and promoting cognate codon recognition, tRNA translocation and recognition by synthetases. We have recently discovered that four essential enzymes, TsaC, TsaB, TsaD and TsaE, are required and sufficient for t6A37 biosynthesis in bacteria. Because the four enzymes are essential in bacteria, and two of them are unique to the bacterial domain, the t6A37 pathway is a compelling potential target for the development of a new generation of anti- bacterial therapeutics. In the proposed work, the catalytic mechanism of the first and universal enzyme in the pathway, TsaC, will be elucidated using a multi-pronged approach comprising kinetic, biochemical, NMR and crystallographic methods. Simultaneously, the precise roles of other three proteins in the biosynthetic process and their concerted interactions with each other and with substrates will be elucidated using a combination of approaches, including transient kinetics experiments, tRNA binding experiments, NMR studies, and X-ray crystallography. This is a multi-institutional collaborative project that summons cross-disciplinary expertise and methodologies to elucidate the mechanistic and structural basis for t6A37 biosynthesis, and establish the experimental foundation for the development of the t6A37 pathway as an antibacterial target.