This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.In research performed at BioCAT we have investigated structural and solvent contributions to RNA folding and stability. Two questions have guided our small-angle x-ray scattering experiments: 1) How dynamic are RNA folding intermediates? 2) What interactions are responsible for maintaining an intermediate conformation? Previous experiments at BioCAT (2004) indicated an intermediate RNA conformation in the background of 0.4 mM MgCl_2 . This year we performed SAXS experiments in the background of various concentrations of monovalent cations. If our RNA folding intermediate is highly dynamic we would anticipate monovalent cations to increase this dynamic nature through shielding of the electrostatic repulsion caused by the negatively charged RNA backbone. Instead we found that this RNA intermediate maintained a constant size and shape as measured by SAXS even upon addition of 500 mM NaCl. This was a very surprising result and suggests that the intermediate conformation is not very dynamic. Additionally, it suggests that electrostatic repulsion does not play a significant role in maintaining this conformation. We then hypothesized that structural interactions in the core of this intermediate were responsible for maintaining the singular nature of this intermediate conformation. We made mutations to disrupt these interactions and then visited BioCAT for follow-up experiments. Indeed this mutated RNA is collapsed relative to the wild-type RNA though the addition of 500 mM NaCl does not collapse it further. These experiments indicate that this distinct RNA folding intermediate is stabilized by particular contacts in the core of the molecule. Manuscript in preparation.
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