Structure and Dynamics of RNA

描述(由申请人提供)：这项建议将继续我们的核磁共振研究核磁共振的RNA结构和动力学的计划。尽管RNA的新的生物学功能不断被发现，但与蛋白质相比，关于RNA的三维结构的信息仍然相对较少。该项目的一个组成部分包括开发用于确定RNA溶液结构的改进的核磁共振方法，包括将TROSY实验应用于大分子的研究。我们还将开发改进的方法，将残留偶极耦合纳入RNA的溶液结构确定中。剩余的偶极耦合数据提供了长程角度信息，补充了标准的短程距离和扭转角核磁共振数据。这种远程结构信息使得极大地提高溶液中核酸的全局结构测定成为可能。此外，还将开发获得附加偶极耦合约束条件的方法，并利用剩余偶极耦合数据改进RNA结构精化。用于确定分子全局结构的剩余偶极耦合方法将应用于一系列tRNA和tRNA类分子。我们将研究一些标准的和不寻常的胞浆和线粒体tRNA的全球构象。其中一些线粒体tRNA突变与多种人类疾病有关，我们将确定导致疾病的突变是否会导致tRNA全球结构的变化。一个常见的RNA三级结构基序-GAAA四元环-受体相互作用的动力学将通过13C驰豫和剩余偶极耦合核磁共振实验来研究。弛豫实验探测RNA的局域动力学，偶极耦合探测RNA的磁区动力学。最近发展起来的单分子荧光共振能量转移技术也将被用于研究这个GAAA-TETRALOOP三级基序中螺旋结构域的动力学。这种核磁共振弛豫、核磁共振偶极耦合和单分子荧光技术的组合将提供广泛时间范围内构象波动的综合图景。这些数据将有助于更好地理解动力学与这个RNA三级结构基序的功能和稳定性之间的关系。

DESCRIPTION (provided by applicant): This proposal will continue our program on NMR studies of RNA structure and dynamics. Although new biological functions for RNAs continue to be discovered, there is still relatively little information on the three-dimensional structures of RNAs, as compared with proteins. One component of this project involves development of improved NMR methods for solution structure determinations of RNA, including application of TROSY experiments for studies of larger molecules. We will also develop improved methods for incorporating residual dipolar couplings into solution structure determinations of RNA. Residual dipolar coupling data provide long-range angle information that complements standard short-range distance and torsion angle NMR data. This long-range structural information makes it possible to dramatically improve the global structure determination of nucleic acids in solution. Methods will also be developed for obtaining additional dipolar couplings constraints and improving the RNA structure refinements with residual dipolar coupling data.The residual dipolar coupling methods for determining global structures of molecules will be applied to a series of tRNA and tRNA-like molecules. We will study the global conformation of a number of standard and unusual cytosolic and mitochondrial tRNAs. Some of these mitochondrial tRNA mutants have been implicated in various human diseases, and we will determine if mutations that cause disease lead to changes in the global structure of the tRNA.The dynamics of a common RNA tertiary structural motif, the GAAA tetraloop-receptor interaction, will be studied by 13C relaxation and residual dipolar coupling NMR experiments. The relaxation experiments probe the local dynamics, and the dipolar couplings probe domain dynamics for the RNA. Recently developed single molecule fluorescence resonance energy transfer techniques will also be used to study dynamics for helical domains in this GAAA-tetraloop tertiary motif. This combination of NMR relaxation, NMR dipolar coupling and single molecule fluorescence techniques will provide a comprehensive picture of the conformational fluctuations over a wide range of timescales. These data will lead to a better understanding of how dynamics correlates with the function and stability of this RNA tertiary structural motif.