NMR STRUCTURE DETERMINATIONS OF RNA

RNA 的 NMR 结构测定

• 批准号: 6169731
• 负责人: ARTHUR PARDI
• 金额: $ 20.91万
• 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/6169731
• 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关于NMR结构研究的计划 RNA。 尽管我们的理解有很大增加 在RNA在单元中执行的各种功能中，仍有 关于RNA的三维结构的相对较少的信息。 该项目的一部分涉及开发改进的NMR方法 用于共振分配和溶液结构RNA的确定 寡核苷酸。 标记的13C/15N产生的能力 定义序列的RNA允许强大的2D，3D和4D 杂核NMR实验适用于核酸。 一些 将作为研究的领域包括：网络编辑的应用 消除某些类型的自旋扩散的噪声实验 文物；改进的方法用于提取耦合常数 同位素标记的RNA;探测二聚/聚合状态 通过NMR的转化扩散测量值的RNA；和 通过使用的方法提高RNA的产量和纯化的方法 核酶作为位点特异性RNA裂解试剂。 13c放松 实验将用于探测几种RNA的动力学 结构基序，包括Cuug RNA四边形。 力量 T1P的依赖性将以1H，13C，15N和31P进行测量 在此四边形中的共鸣，以探测较慢的动作 MUS到MS时尺。 NMR将用于探测结构和 铁响应元件（IRE）RNA的Synalics。 这个RNA图案 位于所有铁蛋白mRNA和 3'转铁蛋白受体mRNA的未翻译区域。 结合 使用铁响应元件结合蛋白，IRE RNA有助于 通过控制铁的翻译来调节细胞中的铁水平 导入和存储蛋白。 多维异核NMR 技术将用于确定三维结构 ire rna已鉴定出的几个突变ire rnas 通过体外选择实验。