Substrate Recognition of a tRNA Modifying Enzyme

描述(申请人提供)：Queuine(约100个天然修饰碱基之一)在真细菌和真核生物的4个tRNA(反密码子枪)的摆动位置(34)中被发现。它是通过tRNA-鸟氨酸转糖基酶(TGT，也在各个王国中保守)催化的转录后碱基交换引入tRNAs的。已有研究表明，TGT是引起痢疾的福氏志贺菌致病所必需的。虽然TGT基因已被证明不是细胞培养中生长所必需的，但TGT与致病性之间的关系尚不清楚，目前的数据表明TGT可能是一个新的抗生素靶点。以往的研究已经阐明了真细菌TGT识别和区分特定tRNA和杂环底物的分子基础的许多方面。然而，要了解真核生物和真细菌TGT的差异特性，仍需要做大量的工作。除了与增殖、分化、逆转录病毒移码和细菌毒力有关外，人们对队列的生理作用知之甚少，更不用说这些作用是如何发挥的了。这项提议有两个首要目标。首先是阐明真细菌TGT与人类TGT的异同。这些研究是合理设计真细菌TGT选择性抑制剂的前提。我们的第二个首要目标是试图阐明队列在真细菌和真核生物中的生理作用(S)。鉴于TGT作为一种新的抗生素靶点的潜力，了解队列的生理作用(S)以及人类和真细菌之间的任何差异是很重要的。为了实现这些目标，我们提出了一些具体的目标，将许多技术和方法(如分子生物学、酶学、结晶学、计算化学、合成化学、基因组学和蛋白质组学)应用到这个问题上。为了以严格的方式进行这些研究，我们与主要专家建立了合作关系，并利用了密歇根大学的现有资源。清楚地了解真细菌和人类TGT之间的异同将具有许多原因，但最明显的是对未来新型抗生素的设计具有重要意义。

DESCRIPTION (provided by applicant): Queuine (one of ca. 100 naturally occurring modified bases) is found in the wobble position (34) of 4 tRNAs (anticodons GUN) in eubacteria and eukaryotes. It is introduced into tRNAs by a post-transcriptional base exchange catalyzed by tRNA-guanine transglycosylase (TGT, also conserved across the kingdoms).. It has been shown that TGT is required for pathogenicity of the dysentery-causing bacterium Shigella flexneri. While the tgt gene has been shown not to be required for growth in cell culture, the relationship between tgt and pathogenicity is not understood, and the present data suggest that TGT could be a novel antibiotic target. Previous studies have elucidated many aspects of the molecular basis of the recognition and discrimination of specific tRNAs and heterocyclic substrates by the eubacterial TGT. However, much work still needs to be done to understand the differential properties of eukaryotic vs. eubacterial TGTs. Other than correlations with proliferation, differentiation, retroviral frameshifting, and bacterial virulence, little is known about the physiological roles of queuine let alone how these roles are carried out. There are two overarching goals of this proposal. The first is to elucidate the similarities and differences between the eubacterial TGT and the human TGT. These studies are prerequisite to the rational design of selective inhibitors of the eubacterial TGT. Our second overarching goal is to try to elucidate the physiological role(s) of queuine in eubacteria and eukaryotes. Given the potential of TGT as a novel antibiotic target, it is important to understand the physiological role(s) of queuine and any differences between human and eubacteria. In order to achieve these goals, we have proposed a number of specific aims that bring many techniques and approaches (e.g., molecular biology, enzymology, crystallography, computational chemistry, synthetic chemistry, genomics, and proteomics) to bear on this problem. To carry out these studies in a rigorous fashion, we have established collaborations with key experts and take advantage of the resources available here at the University of Michigan. A clear understanding of the similarities and differences between the TGTs from eubacteria and human will be of significance for many reasons but most obviously for the future design of novel antibiotics.