AbstractsChemistry

Nucleic acids experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The widespread importance of induced fit and order-disorder transition in nucleic acids recognition by proteins makes it imperative that these motional properties are characterized quantitatively. Until now, very few studies have been dedicated to the systematic characterization of RNA or DNA motion and to their changes upon protein binding. Here we demonstrate that it is possible to characterize the internal dynamics of nucleic acids by 13C relaxation measurements in free RNA, protein-bound RNA and in DNA.In the U1A protein-RNA complex we report 13C NMR relaxation studies of base and ribose dynamics for the free RNA internal loop target of human U1A protein and the 3'UTR RNA-protein complex. We report the quantitative analysis of both fast (ns-ps) and intermediate (mus-ms) motions by measuring 13C T1, T1rho and heteronuclear NOEs for sugar and base nuclei, as well as the power dependence of T1rho at 500 and 750 MHz, and analyze these results using the model-free formalism.The results define a model where the RNA internal loop region 'breathes' on a mus-ms time scale with respect to the double helical regions while the residues at the very tip of the loop undergo faster (ns-ps) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts. Changes in relaxation observed upon complex formation demonstrate that the protein-binding site becomes rigid in the complex, but the upper stem-loop that defines the secondary structure of this RNA experiences unexpected motional freedom. Together with previous studies of the RNA-bound protein, they demonstrate that protein-RNA interfaces experience complex motions that modulate the strength of individual interactions.We have also compare solution and solid state NMR results to probe the internal localized motions of a DNA dodecamer that contains a recognition site for the M.HhaI enzyme. Our results strongly suggest that mus-ns motions contribute to the functionally relevant dynamic properties of nucleic acids during DNA methylation.