This proposal will continue our program on NMR structural studies of RNA. Although there has been a huge increase in our understanding of the various functions that RNAs perform in cells, there is still relatively little information on the three-dimensional structures of RNA. Part of this project involves development of improved NMR methods for resonance assignment and solution structure determinations of RNA oligonucleotides. The ability to generate uniformly 13C/15N labeled RNAs of defined sequence has allowed powerful 2D, 3D and 4D heteronuclear NMR experiments to be applied to nucleic acids. Some areas that will be studies include: application of network-editing NOESY experiments for eliminating certain types of spin-diffusion artifacts; improved methods for extracting coupling constants in isotopically labeled RNAs; probing the dimerization/aggregations state of RNAs by NMR measurements of translational diffusion; and methods for improving the yields and purification of RNAs by use of ribozymes as site-specific RNA cleavage reagents. 13C relaxation experiments will be used to probe the dynamics of several Rna structural motifs, including a CUUG RNA tetraloop. The power dependence of the T1p will be measured for 1H, 13C, 15N and 31P resonances in this tetraloop in order to probe slower motions on the mus to ms timescale. NMR will be used to probe the structure and synamics of the iron response element (IRE) RNA. This RNA motif is located at the 5' untranslated region of all ferritin mRNAs and the 3' untranslated region of transferrin receptor mRNA. In conjunction with the iron response element binding protein, the IRE RNA helps regulate iron levels in a cell by controlling the translation of iron import and storage proteins. Multi-dimensional heteronuclear NMR techniques will be used to determine the three-dimensional structure of the IRE RNA several mutant IRE RNAs that have been identified through in vitro selection experiments.
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