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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。该项目的目标是获得SAXS/WAXS数据，用于对包括HIV-1移码元件（HIV-45），GAAA四环（TECTO 43）和U2-U6四螺旋连接在内的几种RNA进行联合NMR/SAXS高分辨率结构测定。由于缺乏H-H距离限制、共振线加宽和重叠以及难以获得取向RDC限制，高分子量RNA通过溶液NMR进行结构测定仍然极具挑战性。在这方面，SAXS数据代表了理想的互补成分，编码了NMR数据中大部分缺失的长程翻译信息。我们的研究将是第一个高分子量RNA的研究。 我们还将获得TECTO 43的WAXS数据。这种RNA的高度对称性导致其I（q）在1.6-2.0 A-1范围内的特征与连续核苷酸碱基之间的垂直上升之间的直接联系。因此，高q数据的采集将允许直接确定基础上升，这难以仅从NMR数据建立。 HIV-45和TECTO 43的结构NMR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数据从严格持有的子单元组装而成。 我们还将研究RNA的辐射损伤（如果有的话）的程度和性质。将回收样品，并通过溶液NMR研究13 C/1H化学位移的任何位点特异性变化。

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.