TRNA METHYLASE AND TRNA PSEUDOURIDINE SYNTHASE

长期目标是了解催化机制， tRNA（m5 U 54）-甲基转移酶（RUMT）的底物识别。 一 第二个目标是启动tRNA假尿苷合酶I的工作 和II（II，psi 55和I，hisT）。 具体目标概括如下：（1）研究 tRNA和tRNA的T臂结合时发生的构象变化 关于Rumt 将使用停流荧光探测快速动力学 淬火 RNA底物的诱变将旨在破坏 二级和三级RNA结构，以及RUMT对催化的影响 会破坏RNA的二级和三级结构， 将对RUMT的催化作用进行评估。 在适当的合作中，NMR 对酶和底物进行X射线晶体学分析， 单独和复杂。 (2)我们将研究tRNA Rumt的认可。 RNA足迹技术将用于识别 RUMT-RNA接触T臂外。RNA类似物的化学合成 将用于获得具有各种官能团的基底 取代，例如在特定位置的脱氧核糖。 体外 选择（SELEX）将用于鉴定“最佳结合”序列。 （三） 我们将尝试结晶RUMT和RUMT-RNA复合物，用于未来的X射线衍射。 结构测定 （四）、 我们将确定是否RNA以外的 tRNA是RUMT的底物。 (5)研究将使用伪 尿苷（psi 55）合酶II和假尿苷合酶I（his T），密切相关 根据我们提出的RUMT研究的具体目标。 这项工作在生物医学的几个不同层次上都具有重要意义。 research. 首先，该研究旨在更多地了解酶 催化，提供洞察这些反应如何发生在复杂的 RNA分子的环境。 第二，作品试图识别 有助于蛋白质-RNA识别的元件， 某些蛋白质通过这些规则识别 核糖核酸 这项工作还试图确定tRNA的构象变化， 伴随蛋白质识别，并开始对独特的结构研究， RNA-蛋白质复合物。 第三，如果其他RNA是 RUMT（或psi 55合酶），这里进行的诱变研究将 帮助他们识别。 最后，一些抗癌的效果 FUra试剂可能是由于其掺入RNA。 作为这方面的工作 进展，这一点将得到澄清，并可能导致 识别和开发新的药物靶点。

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.