RNA modification: Mechanism and links to other metabolic pathways

摘要 转移RNA(TRNA)是翻译的关键分子，它们能够准确和 有效地解码遗传信息依赖于转录后修饰 核苷酸，特别是在关键的反密码子茎环(ASL)中。这些方面的不足 修饰可能是致命的，并已与各种多效性表型和 人类疾病状态。这项研究计划的长期目标是制定一项详细的 了解复杂tRNA修饰的生物合成途径，这些作用 修饰在细胞生理学中发挥作用，并在其途径中识别新的靶点 治疗性干预。 这一应用特别侧重于阐明形成的分子机制。 和细菌中普遍存在的ASL修饰苏氨酰氨基甲酰腺苷(T6A)的特异性， 并阐明了将其与细菌细胞壁合成联系起来的调控途径。T6A是一种复合体 在解码ANN密码子的tRNA的ASL中发现了修饰，这对tRNA的功能至关重要 通过防止核糖体移码，促进同源密码子识别，促进tRNA 易位，并作为氨基酰-tRNA合成酶的识别决定因素。 在上一个资金阶段，我们就t6A达成了第一个机械性的提案。 蛋白质TsaC2、Tsab和TsaD共同作用的生物合成循环 底物tRNA的A37上的苏氨酰氨基甲酰，而TSAE提供了意想不到的ATPase活性 循环周转所需。我们还发现TSAE是一种新的细菌S/T/Y激酶， 我们的生物信息学分析表明，t6A的生物合成与细胞壁代谢有关， 这对广泛报道的相关细胞壁合成表型提出了新的问题 伴T6A缺乏症。 在当前的申请中，我们提出了4个具体目标，使我们能够阐明1) 细菌t6A系统的tRNA特异性，2)TC-AMP在t6A中的转移机制 生物合成，3)ATP水解酶驱动t6A循环周转的机制，以及4)连接 TSAE与细胞壁合成和/或细胞分裂之间的关系。这项工作将通过以下方式完成 生化、结构、遗传和生理学方法的结合。

Summary Transfer-RNAs (tRNA) are key molecules of translation, and their ability to accurately and efficiently decode genetic information is dependent on post-transcriptional modification of nucleotides, particularly in the critical anticodon stem loop (ASL). Deficiencies in these modifications can be lethal, and have been linked to a variety of pleiotropic phenotypes and human disease states. The long-term goals of this research program are to develop a detailed understanding of the biosynthetic pathways to complex tRNA modifications, the roles these modifications play in cellular physiology, and to identify novel targets in their pathways for therapeutic intervention. This application specifically focuses on elucidating the molecular mechanisms of formation and specificity of the universal ASL modification threonylcarbamoyladenosine (t6A) in bacteria, and elucidating the regulatory pathways that link it to bacterial cell wall synthesis. t6A is a complex modification found in the ASLs of tRNAs decoding ANN codons, and is critical for tRNA function by preventing ribosomal frameshifting, promoting cognate codon recognition, facilitating tRNA translocation, and serving as a recognition determinant for aminoacyl-tRNA synthetases. In the previous funding period we arrived at the first mechanistic proposal for the t6A biosynthesis cycle in which the proteins TsaC2, TsaB and TsaD function together to install threonylcarbamoyl on A37 of substrate tRNA, while TsaE provides an unexpected ATPase activity required for turnover of the cycle. We also discovered that TsaE is a novel bacterial S/T/Y kinase, and our bioinformatic analyses suggested a linkage of t6A biosynthesis to cell wall metabolism, which raises new questions about the widely reported cell wall synthesis phenotypes associated with t6A deficiency. In the current application we propose 4 specific aims that will allow us to elucidate 1) the tRNA specificity of the bacterial t6A system, 2) the mechanism of TC-AMP transfer in t6A biosynthesis, 3) the mechanism of ATP-hydrolysis driven turnover of the t6A cycle, and 4) the links between TsaE and cell-wall synthesis and/or cell division. This work will be accomplished through a combination of biochemical, structural, genetic, and physiologic approaches.