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.
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