STUDY OF RNA STRUCTURE IN SOLUTION BY SAXS AND HIGH-RESOLUTION NMR

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的目标是获得SAXS/WAXS数据，用于联合核磁共振/SAXS高分辨结构测定几个RNA，包括HIV-1移码元件(HIV-45)、GAAA四环(TECTO43)和U2-U6四螺旋连接。由于H-H距离约束的稀缺、共振线的加宽和重叠以及获得定向RDC约束的困难，高分子RNA在通过溶液核磁共振确定结构方面仍然具有极大的挑战性。在这方面，SAXS数据代表了一种理想的补充成分，对核磁共振数据中大部分缺失的长距离翻译信息进行了编码。我们的研究将是第一次针对高相对分子质量的RNA进行此类研究。 我们还将获得TECTO43的WAXS数据。这种RNA的高度对称性导致其在1.6-2.0A-1范围内的I(Q)特征与连续核苷酸碱基之间的垂直上升直接相关。因此，获取高Q数据将允许直接确定碱基上升，这是很难完全从核磁共振数据中确定的。 HIV-45和TECTO43的结构核磁共振约束将与SAXS数据同时拟合，以产生最终模型。U2-U6模型将主要基于SAXS数据由刚性持有的子单元组装而成。 我们还将调查对RNA的辐射损伤(如果有的话)的范围和性质。样品将被回收并通过溶液核磁共振研究13C/1H化学位移的任何特定位置的变化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to acquire SAXS/WAXS data for a joint NMR/SAXS high-resolution structure determination for several RNAs including the HIV-1 frameshift element (HIV-45), the GAAA tetraloop (TECTO43), and the U2-U6 four-helix junction. High-MW RNAs remain extremely challenging for the structure determination via solution NMR due to scarcity of the H-H distance restraints, resonance line broadening and overlap, and difficulties in obtaining the orientational RDC restraints. In that respect, SAXS data represents an ideal complementary component, encoding for the long-range translational information largely missing from the NMR data. Our study will be the first one of this kind for the high-MW RNAs. We will also acquire WAXS data for TECTO43. High symmetry of this RNA results in a direct connection between the features of its I(q) in the 1.6-2.0 A-1 range and the vertical rise between the successive nucleotide bases. Acquisition of high q data will thus allow a direct determination of the base rise which is difficult to establish from the NMR data exclusively. Structural NMR restraints for HIV-45 and TECTO43 will be fitted simultaneously with the SAXS data to yield the final models. U2-U6 model will be assembled from rigidly held subunits based primarily on the SAXS data. We will also investigate the extent and nature of the radiation damage (if any) to the RNAs. The samples will be recovered and studied by the solution NMR for any site-specific changes of the 13C/1H chemical shifts.