Structure and Dynamics of RNA

描述（由申请人提供）：该提案将继续我们的RNA结构和动力学的核磁共振研究项目。尽管rna的新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涉及发展改进的核磁共振方法来确定RNA的溶液结构，包括应用TROSY实验来研究大分子。我们还将开发改进的方法，将残余偶极偶联纳入RNA的溶液结构测定。剩余的偶极耦合数据提供了远程角度信息，补充了标准的短程距离和扭转角核磁共振数据。这种远距离的结构信息使得极大地提高溶液中核酸的整体结构测定成为可能。研究人员还将开发新的方法，以获得额外的偶极偶联约束，并利用剩余的偶极偶联数据改进RNA结构。用于确定分子整体结构的剩余偶极偶联方法将应用于一系列tRNA和tRNA样分子。我们将研究一些标准的和不寻常的细胞质和线粒体trna的整体构象。其中一些线粒体tRNA突变与各种人类疾病有关，我们将确定引起疾病的突变是否会导致tRNA整体结构的变化。通过13C弛豫和残余偶极偶联NMR实验，研究了常见的RNA三级结构基序GAAA四环受体相互作用的动力学。弛豫实验探测RNA的局部动力学，偶极偶联实验探测RNA的结构域动力学。最近发展的单分子荧光共振能量转移技术也将用于研究这种gaaa -四环三级基序的螺旋结构域的动力学。核磁共振弛豫、核磁共振偶极偶联和单分子荧光技术的结合将提供在大范围时间尺度上构象波动的全面图像。这些数据将有助于更好地理解动力学如何与RNA三级结构基序的功能和稳定性相关。

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.