TRNA METHYLASE AND TRNA PSEUDOURIDINE SYNTHASE

TRNA 甲基化酶和 TRNA 假尿苷合酶

• 批准号: 2701625
• 负责人: DANIEL V. SANTI
• 金额: $ 18.49万
• 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/2701625
• 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机制和 tRNA (m5U54)-甲基转移酶 (RUMT) 的底物识别。 一个 次要目标是启动 tRNA 假尿苷合酶 I 的研究 和 II（II、psi55 和 I、hisT）。 具体目标概括如下：（一）研究 tRNA 和 tRNA T 臂结合时发生的构象变化 到鲁姆特。 将使用停流荧光来探测快速动力学 淬火。 RNA底物的诱变旨在破坏稳定性 RNA二级和三级结构以及RUMT对催化的影响 会破坏二级和三级RNA结构的稳定性，并且效果 将评估 RUMT 的催化作用。 在适当的合作中，NMR 并对酶和底物进行 X 射线晶体学分析， 单独的和复杂的。 (2)我们将研究tRNA的各个方面 获得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官能团的底物 取代，例如特定位置的脱氧核糖。 体外 选择 (SELEX) 将用于识别“最佳结合”序列。(3) 我们将尝试结晶 RUMT 和 RUMT-RNA 复合物以用于未来的 X 射线 结构测定。 （4）。 我们将确定是否存在除 tRNA 是 RUMT 的底物。 (5) 研究将用伪进行 尿苷 (psi55) 合酶 II 和伪尿苷合酶 I (his T) 密切相关 遵循我们提出的 RUMT 研究的具体目标。 这项工作在生物医学的几个不同层面都具有重要意义 研究。 首先，该研究旨在更多地了解酶 催化，深入了解此类反应如何在复杂的环境中发生 RNA分子的环境。 其次，这项工作试图确定 有助于蛋白质-RNA 识别并揭示一般性的元素 某些蛋白质识别共同结构特征的规则 核糖核酸。 这项工作还试图识别 tRNA 的构象变化，这些变化 伴随蛋白质识别，并启动独特的结构研究 RNA-蛋白质复合物。 第三，如果其他 RNA 是潜在的底物 RUMT（或 psi 55 合酶），此处进行的诱变研究将 协助他们进行身份识别。 最后说一下抗癌的一些作用 FUra 剂可能是由于其掺入 RNA 所致。 作为该领域的工作 随着进展，这一点将得到澄清，并可能导致 新药物靶标的识别和开发。