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，其准确有效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。这些修饰中的缺陷导致遗传性人类线粒体疾病，并且修饰的核苷用作敏感的诊断剂，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进行治疗干预。本申请特别旨在从生物化学和结构上阐明通用修饰的核苷苏氨酰氨基甲酰基腺苷（t6A37）生物合成的分子机制。t6A37是ASL的一种复杂的、古老的修饰，在所有生命形式中的所有解码ANN密码子的tRNA中发现。它通过阻止核糖体移码和促进同源密码子识别、tRNA移位和合成酶识别而对tRNA功能至关重要。我们最近发现，四种必需的酶，TsaC，TsaB，TsaD和TsaE，是细菌中t6A37生物合成所必需的。由于这四种酶在细菌中是必需的，并且其中两种是细菌结构域所特有的，因此t6A37途径是开发新一代抗菌治疗剂的引人注目的潜在靶标。在拟议的工作中，第一个和通用酶的催化机制的途径，TsaC，将阐明使用多管齐下的方法，包括动力学，生物化学，NMR和结晶学方法。同时，其他三种蛋白质在生物合成过程中的确切作用及其相互之间和与底物的协同作用将使用多种方法的组合来阐明，包括瞬态动力学实验，tRNA结合实验，NMR研究和X射线晶体学。这是一个多机构的合作项目，召集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，并为开发t6A37途径作为抗菌靶标建立实验基础。

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.