Many RNAs and DNAs function by undergoing significant conformational changes when binding to proteins or small molecules. In order to understand how motions contribute to RNA and DNA function, static structural data obtained by crystallography and NMR must be augmented by analysis of the motions associated with functionally relevant structural changes. These dynamic studies must establish the rates at which such changes occur as well as the amplitudes and precise atomic nature of any intrinsic motion present in the nucleic acids. Yet biological dynamics is complex and spans at least twelve orders of magnitude in rates, requiring multiple spectroscopic techniques to be applied in a concerted fashion. How to best obtain this information is the focus of this proposal. In the previous funding period we have developed and applied new solid state NMR methods, computational modeling techniques and ultrafast spectroscopic methods. In two independent technical breakthroughs, we have prepared solid state NMR samples with sufficient resolution to record multidimensional solid state 13C spectra of RNA and have recorded 2-dimensional solution NMR spectra with resolution of just a few seconds to follow conformational changes in riboswitches in real time. By introducing multidimensional, magic angle spinning (MAS) solid state NMR techniques to nucleic acids;applying real-time NMR methods to RNAs with time resolution of a few seconds;merging experimental NMR results with long time scale molecular modeling techniques, we will provide unprecedented insight into molecular motions in three paradigmatic nucleic acid systems by: 1. Conclusively proving (or disproving) whether intrinsic motions that partially pre-extrude a DNA base from the Watson-Crick paired double helix provide a pathway for the extrusion of the cytosine that is methylated by the HhaI methylase enzyme. 2. Examining whether a very common protein-binding signal in RNA, a single-stranded bulge interrupting two helices as found in K-turn RNAs, fluctuates transiently through a highly kinked state that is already pre-disposed for binding of its cognate protein. 3. Following the ligand-induced conformational change in an adenine-sensing riboswitch by using real time NMR. With resolution of a few seconds, to record chemical shift, residual dipolar couplings and paramagnetic enhancements to establish the structure of intermediates along the pathway and the trajectory linking the two RNA conformations. By executing this technically ambitious program, we will provide new insight into the dynamics underpinning of RNA and DNA function, and further develop spectroscopic and computational techniques that will be widely applicable to other nucleic acids.
Many RNA and DNA molecules perform their biological functions by altering their conformation in response to biological signals, namely proteins or small molecules that bind to them. By combining magnetic resonance spectroscopies and developing new spectroscopic and computational methods, this project will examine how RNA and DNA motions occur in paradigmatic systems and study how dynamics contribute to their biological function.
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