ENZYME MEDIATED THIOL MODIFICATION OF TRNA

tRNA中的碱基修饰代表了一个关键的 细胞代谢过程-信使的忠实翻译 RNA转化为蛋白质 修饰酶的独特之处在于它们的 底物是高度结构化的tRNA分子;因此，它们提供了一种 研究蛋白质-RNA相互作用的机会， 机械酶学 这项工作的长期目标是 了解巯基修饰的酶机制， E. coli tRNA、尿苷8（U8）和腺苷37（A37）， 与细菌代谢中的调节事件有关。 在 此外，这些酶将用于探索RNA-蛋白质相互作用 为了理解它们所观察到的特异性的基础， 核苷酸水平。 具体工作目标如下：（1）酶 将开发分析以允许修饰的纯化 内切酶 酶将被纯化至同质，使用亲和层析。 来自底物的固定化RNA片段的层析 特异性研究。 将从E.杆菌 使用来自纯化蛋白质的序列信息。 (2)的 将研究硫转移到 tRNA碱基，在每种情况下通过不同的辅因子来自半胱氨酸， 可能是不同的机制。 (3)铁依赖在糖尿病中的作用 A37的硫醇化将通过开发特定的 这一步的抑制剂和观察这种代谢作用 与先前描述的遗传突变体相比，抑制。 (4)的 酶的tRNA底物特异性将使用以下文献阐明： 体外选择技术，探索小学，中学和 作为底物活性的三级结构决定因素。 硫醇 修饰将被用作化学标签来检索活性分子 从E. coli tRNAPhe。 与医疗问题的具体相关性在于，A37 修饰被认为是细菌中的一种传感器， 低铁可用性。 这些条件出现在宿主中（例如， 哺乳动物）肠道致病菌所居住的液体。 在 此外，从这项工作中获得的知识直接关系到 目前对RNA和RNA之间的分子识别事件的研究， 蛋白质，如逆转录病毒组装和RNA加工和运输， 参与了许多疾病相关的过程。

Base modification in tRNA represents the find tuning of a crucial process in cellular metabolism - the faithful translation of messenger RNA into protein. The modification enzymes are unique in that their substrates are highly structured tRNA molecules; thus, they provide an opportunity to study protein-RNA interactions in the context of mechanistic enzymology. The long range goal of this work is to understand the enzymatic mechanisms of thiol modification at two specific sites in E. coli tRNA, uridine 8 (U8) and adenosine 37 (A37), that are implicated in regulatory events in bacterial metabolism. In addition, these enzymes will be used to explore RNA-protein interactions in an effort to understand the basis for their observed specificity at the nucleotide level. The specific aims of the proposed work are as follows: (1) Enzyme assays will be developed to allow the purification of the modification enzymes. The enzymes will be purified to homogeneity, using affinity chromatography of immobilized RNA fragments derived from substrate specificity studies. The structural genes will be cloned from E. coli using sequence information from the purified proteins. (2) the mechanism will be investigated by which sulfur is transferred to the tRNA bases, in each case from cysteine via different cofactors and presumably different mechanisms. (3) The role of iron dependence in the thiolation of A37 will be explored through the development of specific inhibitors of this step and observation of the metabolic effects of such inhibition as compared to previously described genetic mutants. (4) The tRNA substrate specificity of the enzymes will be elucidated using in vitro selection techniques that explore the primary, secondary and tertiary structure determinants for activity as a substrate. The thiol modifications will be used as chemical tags to retrieve active molecules from mutagenized libraries of E. coli tRNAPhe. Specific relevance to medical issues lies in the fact that the A37 modification is thought to act as a sensor in bacteria for conditions of low iron availability. Such conditions arise in host (e.g., mammalian) fluids inhabited by enteric pathogenic bacteria. In addition, the knowledge gained from this work is directly relevant to the current study of molecular recognition events between RNA and proteins, such as retroviral assembly and RNA processing and transport, that are involved in many disease related processes.