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.
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