RNA modification: Mechanism and links to other metabolic pathways

总结 转移RNA（transfer-RNA，tRNA）是翻译的关键分子，其准确、 有效地解码遗传信息依赖于转录后修饰， 在关键的反密码子茎环（ASL）中，防御在这些 修饰可以是致命的，并且已经与多种多效性表型相关联， 人类疾病状态。这项研究计划的长期目标是制定一个详细的 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，并确定其途径中的新靶点， 治疗干预 本申请特别侧重于阐明形成的分子机制 和细菌中通用ASL修饰苏氨酰氨基甲酰基腺苷（t6 A）的特异性， 并阐明了将其与细菌细胞壁合成联系起来的调节途径。t6 A是一种复合物 在解码ANN密码子的tRNA的ASL中发现的修饰，并且对tRNA功能至关重要 通过阻止核糖体移码，促进同源密码子识别， 易位，并作为氨酰-tRNA合成酶的识别决定簇。 在上一个资助期，我们提出了t6 A的第一个机械提案 蛋白质TsaC 2、TsaB和TsaD共同发挥功能， 苏氨酰氨基甲酰基作用于底物tRNA的A37，而TsaE提供了意想不到的ATP酶活性 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t6 A生物合成与细胞壁代谢有关， 这对广泛报道的细胞壁合成表型提出了新的问题， t6 a缺乏症 在本申请中，我们提出了4个具体目标，这将允许我们阐明：1） 细菌t6 A系统的tRNA特异性; 2）TC-AMP在t6 A中的转运机制 生物合成，3）ATP水解驱动t6 A循环周转的机制，以及4） 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 生物化学、结构、遗传和生理学方法的组合。

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.