Molecular basis for the activity of the Box C/D snoRNP methylation enzyme. A combined solution-state NMR, solid-sate NMR and electron microscopy approach

Box C/D snoRNP 甲基化酶活性的分子基础。

• 批准号: 154387182
• 负责人: Professorin Dr. Teresa Carlomagno
• 金额: --
• 依托单位: School of Biosciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/154387182?language=en
• 关键词: Molecular; basis; activity; Box; snoRNP

Post-transcriptional modifications of both rRNA and mRNA are essential to ensure the correct functioning of several cellular machineries and regulation mechanisms. One of the most important of such post-transcriptional modifications is the methylation of the RNA at the 2’-OH position. The enzyme responsible for RNA methylation is situated in the nucleolus and is assembled around snoRNAs containing a box C/D motif. Three proteins constitute this enzyme in archea, the L7ae, which recognizes first the boxC/D RNA motif, the Nop5 protein and the fibrillarin, which carries out the methylation. Despite several years of biochemical investigation and of crystallization trials, the structure and the functional mechanisms of this fundamental cellular enzyme are still unknown. In this proposal we suggest an interdisciplinary approach to address the structural basis of the catalytic activity of the box C/D snoRNP using a combination of solution-state NMR, solid-state NMR, single particle cryo-electron microscopy, small-angle neutron scattering and molecular modelling. In addition to providing information on the mechanisms of the 2’-OH methylation reaction, this project breaks the traditional barriers among different structural biology techniques and aims to open a new way to the determination of complex structures when traditional crystallographic analysis is not applicable.

rRNA 和 mRNA 的转录后修饰对于确保多种细胞机器和调节机制的正确运作至关重要。这种转录后修饰中最重要的修饰之一是 RNA 2’-OH 位置的甲基化。负责 RNA 甲基化的酶位于核仁中，并围绕含有盒 C/D 基序的 snoRNA 进行组装。古细菌中的这种酶由三种蛋白质组成：L7ae，它首先识别 boxC/D RNA 基序、Nop5 蛋白和原纤维蛋白，它进行甲基化。尽管经过多年的生化研究和结晶试验，这种基本细胞酶的结构和功能机制仍然未知。在本提案中，我们建议采用跨学科方法，结合溶液态 NMR、固态 NMR、单粒子冷冻电子显微镜、小角度中子散射和分子建模来解决盒 C/D snoRNP 催化活性的结构基础。除了提供2’-OH甲基化反应机制的信息外，该项目还打破了不同结构生物学技术之间的传统障碍，旨在为传统晶体学分析不适用时复杂结构的测定开辟一条新途径。