TRNA METHYLASE AND TRNA PSEUDOURIDINE SYNTHASE

TRNA 甲基化酶和 TRNA 假尿苷合酶

• 批准号: 2910150
• 负责人: DANIEL V. SANTI
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910150
• 关键词: RNA methylation; S adenosylmethionine; X ray crystallography; chemical binding; chemical kinetics; cofactor; conformation; crystallization; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; halogenation; methyltransferase; mutant; nuclear magnetic resonance spectroscopy; nucleic acid structure; nucleotide analog; posttranscriptional RNA processing; site directed mutagenesis; stop flow technique; transfer RNA; transmethylation; uracil nucleoside

The long term objective is to understand the mechanism of catalysis and substrate recognition of tRNA (m5U54)-methyltransferase (RUMT). A secondary objective is to initiate work on tRNA pseudo uridine synthase I and II (II, psi55 and I, hisT). The specific aims are summarized as follows: (1) We will study the conformational changes that occur in tRNA and the T arm of tRNA on binding to RUMT. Rapid kinetics will be probed using stopped flow fluorescence quenching. Mutagenesis of the RNA substrate will be aimed at destabilizing secondary and tertiary RNA structure, and the effect on catalysis by RUMT will be destabilizing secondary and tertiary RNA structure, and the effect on catalysis by RUMT will be assessed. In appropriate collaborations, NMR and X-ray crystallography will be performed on the enzyme and substrate, individually and in complex. (2) We will study aspects of tRNA recognition by RUMT. RNA footprinting techniques will be used to identify RUMT-RNA contacts outside of the T arm. Chemical synthesis of RNA analogs will be used to obtain substrates with various functional group substitutions, such as deoxyribose at specific positions. In vitro selection (SELEX) will be used to identify "best binding' sequences. (3) We will attempt to crystallize RUMT and RUMT-RNA complexes for future X-Ray structure determination. (4). We will determine whether RNAs other than tRNA are substrates for RUMT. (5) Studies will be performed with Pseudo Uridine (psi55) Synthase II and Pseudo Uridine Synthase I (his T), closely following the specific aims of our proposed studies of RUMT. This work is significant at several different levels of biomedical research. First, the research seeks to understand more about enzyme catalysis, providing insight into how such reactions occur in the complex environment of an RNA molecule. Second, the work attempts to identify elements contributing to protein-RNA recognition and to uncover general rules by which certain proteins recognize common structural features of RNA. The work also seeks to identify conformational changes of tRNA which accompany protein recognition, and to initiate structural studies on unique RNA-protein complexes. Third, if other RNAs are potential substrates for RUMT (or psi 55 synthase), the mutagenesis studies performed here will assist in their identification. Finally, some effects of the anti-cancer agent FUra may be due to its incorporation into RNA. As work in this area progresses, this point will become clarified and could lead to the identification and exploitation of new drug targets.

长期目标是了解催化和 tRNA（M5U54） - 甲基转移酶（RUMT）的底物识别。 一个 次要目标是启动对tRNA伪尿苷合酶I的工作 和II（II，PSI55和I，Hist）。 具体目的总结如下：（1）我们将研究 tRNA中发生的构象变化和结合tRNA的t臂 到Rumt。 快速动力学将使用停止流量荧光进行探测 淬火。 RNA底物的诱变将旨在破坏稳定 二级和第三级RNA结构，以及对Rumt催化的影响 将破坏次级和三级RNA结构的稳定，效果 关于Rumt的催化，将评估。 在适当的合作中，NMR X射线晶体学将在酶和底物上进行， 单独和复杂。 （2）我们将研究tRNA的各个方面 Rumt的认可。 RNA足迹技术将用于识别 rumt-rna接触T臂外部。 RNA类似物的化学合成 将用于获得具有各种功能组的底物 取代，例如在特定位置的脱氧核糖。 体外 选择（SELEX）将用于识别“最佳绑定”序列。（3） 我们将尝试将Rumt和Rumt-RNA综合体结晶为未来的X射线 结构确定。 （4）。 我们将确定除了RNA以外的其他RNA是否 tRNA是Rumt的底物。 （5）将使用伪进行研究 尿苷（PSI55）合酶II和伪尿苷合酶I（他的T），紧密 遵循我们提议的Rumt研究的具体目的。 这项工作在几个不同水平的生物医学上都很重要 研究。 首先，研究试图更多地了解酶 催化，提供有关这种反应如何发生在复合物中的信息 RNA分子的环境。 其次，工作试图识别 有助于蛋白-RNA识别并发现一般的元素 某些蛋白质识别共同结构特征的规则 RNA。 这项工作还旨在确定tRNA的构象变化， 伴随蛋白质识别，并启动有关独特的结构研究 RNA蛋白质复合物。 第三，如果其他RNA是潜在的基板 RUMT（或PSI 55合酶），此处进行的诱变研究将 协助他们的身份证明。 最后，反癌的某些影响 Fura可能是由于其掺入RNA所致。 作为该领域的工作 进展，这一点将被澄清，并可能导致 识别和剥削新药物靶标。