Recognition and Mechanisms of Transfer RNA Modifications

描述(由申请人提供)：转移RNA(TRNA)执行中央 细胞中的功能及其活动取决于是否存在关键的 它们结构中的修饰核苷酸。出于这个原因，细胞致力于 众多成分(修饰酶及其调节剂)和显著 修饰碱基和核糖的能量(RNA修饰)。两个重要的因素 将尿嘧啶(U)修饰为假尿苷(？F？)55位异构化 和在tRNA的34位被队列(0)取代的鸟嘌呤(G)是 这是本研究的重点。这些反应是由假性尿苷55催化的 合酶(W55S)和tRNA-鸟氨酸转糖基酶(TGT)。在这 提案中，我们将解决与分子识别和 这两种改性反应的机理。 提出了碱基和tRNA底物的各种改变。这些修改后的 底物将通过化学合成来冻结或捕获 酶tRNA在反应途径的不同阶段的中间产物。 生化分析的常规技术，如电泳法 迁移率变化分析、层析和光谱方法将是 用于描述冻结或捕获过程的特征。一旦失速 或者是被捕获的酶。TRNA复合体的获得，将进行结晶 以获得这些络合物的结构信息。 尽管还没有对tRNA 55位的W进行欠修饰 与疾病直接相关，基因的缺失和突变 相应的人类假尿苷合成酶，被称为dyskerin，它是强烈的 核糖体RNA与psi55S同源但参与U到psi异构化 (RRNA)，导致先天性角化不良(DKC)，这是一种罕见的遗传性疾病。病人 患有这种疾病的人患某些疾病的风险随着年龄的增加而增加 癌症，如皮肤和胃肠道的上皮性肿瘤。在……上面 另一方面，Q修饰不足的程度与 人类白血病、淋巴瘤和肺肿瘤的形态特征。 这里提出的研究将揭示分子识别的基础 并对修饰反应的机理进行了探讨。 这些结果可能有助于我们理解可能的 这些疾病的分子病因。

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.