Substrate Recognition of a tRNA Modifying Enzyme

描述（由申请人提供）：马槟榔碱（ca. 100个天然存在的修饰碱基）在真细菌和真核生物中的4个tRNA（反密码子GUN）的摆动位置（34）中发现。它通过由tRNA-鸟嘌呤转糖基酶（TGT，也在各王国中保守）催化的转录后碱基交换被引入tRNA。已经表明，TGT是致病性细菌福氏志贺菌的致病性所必需的。虽然tgt基因已被证明是不需要在细胞培养中的生长，tgt和致病性之间的关系还不清楚，目前的数据表明，TGT可能是一种新的抗生素的目标。以前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，仍然需要做大量的工作来了解真核生物与真细菌TGT的差异特性。 除了与增殖、分化、逆转录病毒移码和细菌毒力的相关性之外，对马槟榔碱的生理作用知之甚少，更不用说这些作用是如何进行的了。这项建议有两个首要目标。首先是阐明真细菌TGT和人类TGT之间的相似性和差异。这些研究是合理设计真细菌TGT选择性抑制剂的先决条件。我们的第二个总体目标是试图阐明在真细菌和真核生物中的生理作用。鉴于TGT作为新型抗生素靶标的潜力，重要的是要了解甜菜碱的生理作用以及人类和真细菌之间的任何差异。为了实现这些目标，我们提出了一些具体的目标，带来了许多技术和方法（例如，分子生物学、酶学、晶体学、计算化学、合成化学、基因组学和蛋白质组学）来解决这个问题。为了以严格的方式开展这些研究，我们与主要专家建立了合作关系，并利用密歇根大学的资源。清楚地了解真细菌和人类TGT之间的相似性和差异性将具有重要意义，原因有很多，但最明显的是未来新型抗生素的设计。

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.