TRNA METHYLASE AND TRNA PSEUDOURIDINE SYNTHASE

长期的目标是了解催化和 TRNA(M5U54)甲基转移酶(RUMT)的底物识别。一个 次要目标是启动tRNA伪尿苷合成酶i的研究工作 和II(II，PSI55和I，HIST)。 具体目标概括如下：(1)我们将研究 TRNA及其T臂结合时的构象变化 敬RUMT。将使用停流荧光来探索快速动力学 淬火。RNA底物的突变将旨在破坏稳定 二级和三级RNA结构及其对RUMT催化作用的影响 会不稳定的二级和三级RNA结构，以及 将对RUMT的催化作用进行评估。在适当的合作中，核磁共振 并对酶和底物进行X射线结晶学检查， 既有个体的，也有复杂的。(2)我们将研究tRNA的各个方面 得到RUMT的认可。将使用RNA足迹技术来识别 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，如特定位置的脱氧核糖。离体 选择(SELEX)将被用来识别“最佳结合”序列。 我们将尝试结晶RUMT和RUMT-RNA络合物以用于未来的X射线 结构确定。(4)。我们将确定其他RNA是否 TRNA是RUMT的底物。(5)将使用PUSIC进行研究 尿苷(PSI55)合成酶II和伪尿苷合成酶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上都具有重要意义 研究。首先，这项研究试图更多地了解酶 催化，提供了对这些反应如何在络合物中发生的洞察 核糖核酸分子的环境。其次，这部作品试图识别 有助于蛋白质-RNA识别和揭示一般情况的元件 某些蛋白质识别共同结构特征的规则 核糖核酸。这项工作还试图确定tRNA的构象变化 伴随蛋白质识别，并启动独特的结构研究 RNA-蛋白质复合体。第三，如果其他RNA是潜在的底物 RUMT(或psi 55合成酶)，这里进行的诱变研究将 协助确认他们的身份。最后，还有一些抗癌的作用。 Fura试剂可能是由于它被整合到RNA中。作为这一领域的工作 进展，这一点将得到澄清，并可能导致 确定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.