Recognition and Mechanisms of Transfer RNA Modifications

描述（由申请人提供）：转移RNA（tRNA）执行中心 细胞中的功能及其活动取决于关键的 在其结构中修饰核苷酸。因此，细胞 许多组分（修饰酶及其调节剂）和重要的 能量修饰碱基和核糖（RNA修饰）。两个重要 修饰，尿嘧啶（U）假尿苷（？f？）55位异构化 以及在tRNA的34位用鸟嘌呤（0）取代鸟嘌呤（G）， 本研究的重点。这些反应由假尿苷55催化 合成酶（W55 S）和tRNA-鸟嘌呤转糖基酶（TGT）。在这 建议，我们将解决有关的问题，分子识别和 这两种反应的机理。 提出了碱基和tRNA底物的各种改变。这些改性 基质将被化学合成，以便冷冻或捕获 在反应途径的沿着不同阶段，tRNA是一种中间产物。 用于生物化学测定的常规技术，例如电泳技术， 迁移率变动分析，色谱和光谱方法，将是 用于表征冷冻或捕获过程。一旦失速 或被困酶。得到tRNA复合物，进行结晶 以获得这些复合物的结构信息。 尽管tRNA的55位W的修饰不足尚未被发现， 与疾病直接相关的是， 相应的人假尿苷合酶，被称为dyskerin，这是强烈的 与psi 55 S同源但参与核糖体RNA中U到psi异构化 （rRNA），导致先天性角化不良（DKC），一种罕见的遗传性疾病。患者 患有这种疾病的人，患上某些疾病的风险会随着年龄的增长而增加。 癌症，如皮肤和胃肠道的上皮肿瘤。对 另一方面，Q欠修饰的程度与 人白血病、淋巴瘤和肺肿瘤的形态学表征。 本文提出的研究将揭示分子识别的基础， 通过这些酶和探针的修饰反应的机制的tRNA。 这些结果可能有助于我们理解 这些疾病的分子原因。

DESCRIPTION (provided by applicant): Transfer RNAs (tRNAs) perform central functions in the cell and their activities depend on the presence of critical modified nucleotides in their structures. For this reason, cells devote numerous components (modification enzymes and their regulators) and significant energy to modify the bases and the riboses (RNA modifications). Two important modifications, uracil (U) to pseudouridine (?f?) isomerization at position 55 and substitution of guanine (G) by queuine (0) at position 34 of a tRNA, are the focus of this study. These reactions are catalyzed by pseudouridine 55 synthase (W55S) and tRNA-guanine transglycosylase (TGT), respectively. In this proposal, we will address issues related to the molecular recognition and mechanisms of these two modification reactions. Various alterations in bases and tRNA substrates are proposed. These modified substrates will be chemically synthesized in order to freeze or trap enzyme.tRNA intermediates at different stages along a reaction pathway. Conventional techniques for biochemical assays, such as electrophoretic mobility shift assay, chromatographic and spectroscopic methods, will be applied to characterize the freezing or trapping processes. As soon as stalled or trapped enzyme. tRNA complexes are obtained, crystallization will be carried out in order to obtain structural information of these complexes. Although under-modification of W at position 55 of a tRNA has not yet been directly correlated to disease, the deletions and mutations of the corresponding human pseudouridine synthase, dubbed dyskerin, which is strongly homologous to psi55S but involved in U to psi isomerization in ribosome RNA (rRNA), causes dykeratosis congenita (DKC), a rare inherited disorder. Patients with this disease have an age-dependent increase in risk for developing certain cancers, such as epithelial tumors of the skin and gastrointestinal tract. On the other hand, the extent of Q under-modification was correlated with morphological characterization of human leukemia, lymphoma, and lung tumors. The studies proposed here will reveal the basis for molecular recognition of tRNAs by these enzymes and probe the mechanisms of the modification reactions. The results will likely contribute to our understanding of the possible molecular causes of these diseases.