NMR STRUCTURE DETERMINATIONS OF RNA

RNA 的 NMR 结构测定

• 批准号: 2003843
• 负责人: ARTHUR PARDI
• 金额: $ 21.76万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2003843
• 关键词: animal genetic material tag; genetic regulatory element; high performance liquid chromatography; human genetic material tag; messenger RNA; nuclear magnetic resonance spectroscopy; nucleic acid structure; oligonucleotides; ribozymes

This proposal will continue our program on NMR structural studies of RNA. Although there has been a huge increase in our understanding of the various functions that RNAs perform in cells, there is still relatively little information on the three-dimensional structures of RNA. Part of this project involves development of improved NMR methods for resonance assignment and solution structure determinations of RNA oligonucleotides. The ability to generate uniformly 13C/15N labeled RNAs of defined sequence has allowed powerful 2D, 3D and 4D heteronuclear NMR experiments to be applied to nucleic acids. Some areas that will be studies include: application of network-editing NOESY experiments for eliminating certain types of spin-diffusion artifacts; improved methods for extracting coupling constants in isotopically labeled RNAs; probing the dimerization/aggregations state of RNAs by NMR measurements of translational diffusion; and methods for improving the yields and purification of RNAs by use of ribozymes as site-specific RNA cleavage reagents. 13C relaxation experiments will be used to probe the dynamics of several Rna structural motifs, including a CUUG RNA tetraloop. The power dependence of the T1p will be measured for 1H, 13C, 15N and 31P resonances in this tetraloop in order to probe slower motions on the mus to ms timescale. NMR will be used to probe the structure and synamics of the iron response element (IRE) RNA. This RNA motif is located at the 5' untranslated region of all ferritin mRNAs and the 3' untranslated region of transferrin receptor mRNA. In conjunction with the iron response element binding protein, the IRE RNA helps regulate iron levels in a cell by controlling the translation of iron import and storage proteins. Multi-dimensional heteronuclear NMR techniques will be used to determine the three-dimensional structure of the IRE RNA several mutant IRE RNAs that have been identified through in vitro selection experiments.

该提案将继续我们的核磁共振结构研究计划 核糖核酸。 虽然我们的认识有了很大的提高 RNA 在细胞中执行的各种功能中，仍然存在 关于RNA三维结构的信息相对较少。 该项目的一部分涉及改进核磁共振方法的开发 用于 RNA 的共振分配和溶液结构测定 寡核苷酸。 能够生成统一的 13C/15N 标记 确定序列的 RNA 允许强大的 2D、3D 和 4D 应用于核酸的异核核磁共振实验。 一些 将研究的领域包括： 网络编辑的应用 用于消除某些类型的自旋扩散的 NOESY 实验 文物；改进的耦合常数提取方法 同位素标记的 RNA；探测二聚/聚集状态 通过平移扩散的 NMR 测量来分析 RNA；和 提高 RNA 产量和纯化的方法 核酶作为位点特异性 RNA 切割试剂。 13C松弛 实验将用于探测几种RNA的动态 结构基序，包括 CUUG RNA 四环。 电源 将测量 1H、13C、15N 和 31P 的 T1p 依赖性 在这个四环中共振，以探测较慢的运动 mus 到 ms 时间刻度。 NMR将用于探测结构和 铁反应元件 (IRE) RNA 的同义词。 这个RNA基序 位于所有铁蛋白 mRNA 的 5' 非翻译区， 转铁蛋白受体 mRNA 的 3' 非翻译区。 结合 IRE RNA 与铁反应元件结合蛋白一起帮助 通过控制铁的翻译来调节细胞中的铁水平 导入和储存蛋白质。 多维异核核磁共振 技术将用于确定三维结构 IRE RNA 已鉴定的几种突变 IRE RNA 通过体外选择实验。