Complex modifications of tRNA: regulatory roles and crosstalk with DNA metabolism

 描述(由申请人提供)：反密码子茎环(ASL)对于解码tRNA的特性至关重要。通用的苏氨酰氨基甲酰腺苷(T6A)和广泛存在的7-去氮鸟苷衍生物Queuosine(Q)分别是第37和34位的两个ASL修饰。我们发现了这两种缺失了几十年的复杂修饰的生物合成途径，为研究这些修饰在体内的作用开辟了道路。我们研究的长期目标是扩大关于复杂tRNA修饰和相关分子的合成和功能的基础知识，并了解它们在核心细胞过程和生理中的作用。目前的应用主要集中在t6A和Q在体内的作用，因为它们的途径复杂，依赖于初级代谢物，而且这两种修饰在解码过程中都起着核心作用，t6A和Q是整合新陈代谢和翻译的理想分子，可能起到不可预见的调节作用。初步结果表明，缺乏t6A会在酵母和细菌中触发未折叠的蛋白质反应，而t6A会影响特定蛋白质的翻译，这将是目标1的重点。初步的表型筛选显示，缺乏Q的大肠杆菌突变株具有金属敏感性或抗性表型，目标2将探索这种修饰在细胞感知和适应金属压力中的作用。 此外，最近的研究表明，RNA和DNA修饰途径的共同点比之前预期的要多得多。我们的意外发现，Q前体被插入到DNA中，这表明在DNA代谢中已经招募了Q合成酶的Paralog，这将在Aim 3中进行研究。Aim 4将专注于T6A合成的第一个酶的Paralog在DNA修复中的潜在作用。这种方法是创新的，因为使用了比较基因组学方法来指导实验工作，这项工作揭示了将新陈代谢和翻译联系起来的新调控机制，以及全新的DNA修饰。这项拟议的研究具有重要意义，因为它将促进我们对关键的tRNA修饰和新的DNA修饰在基础细胞生理学中的作用的理解。

 DESCRIPTION (provided by applicant): The anticodon stem loop (ASL) is critical for decoding properties of tRNA. The universal threonylcarbamoyladenosine (t6A) and the widespread 7-deazaguanosine derivative queuosine (Q) are two ASL modifications at positions 37 and 34, respectively. Our discovery of the biosynthesis pathways for these two complex modifications that had been "missing" for decades has opened the path to study the role of these modifications in vivo. The long-term goal of our research is to expand fundamental knowledge on the synthesis and function of complex tRNA modifications and related molecules, and to understand their roles in core cellular processes and physiology. The current application focuses on the in vivo role of t6A and Q. Because their pathways are complex and draw on primary metabolites and both these modifications have central roles in decoding, t6A and Q are ideal candidates for molecules that integrate metabolism and translation and could play unforeseen regulatory roles. Preliminary results suggest that the absence of t6A triggers an unfolded protein response in both yeast and Bacteria and that t6A affects the translation of specific proteins and this will be the focus of Aim 1. Preliminary phenotypic screens show that Escherichia coli mutants that lack Q have metal sensitivity or resistance phenotypes, and the role of this modification in how cells sense and adapt to metal stresses will be explored in Aim 2. In addition, it has recently been shown that RNA and DNA modifications pathways have much more in common than previously anticipated. Our unexpected discovery that Q precursors are inserted in DNA suggests that paralogs of Q synthesis enzymes have been recruited in DNA metabolism and this will be studied in Aim 3. Aim 4 will focus on the potential role in DNA repair of a paralog of the first enzyme of t6A synthesis. The approach is innovative because comparative genomic methods were used to guide the experimental effort, and this work is revealing new regulatory mechanisms linking metabolism and translation as well totally novel modifications of DNA. The proposed research is significant because it will advance our understanding of the role of critical tRNA modifications and novel DNA modifications in fundamental cellular physiology.