Time-resolved hydroxyl radical footprinting

Time-resolved synchrotron x-ray hydroxyl radical footprinting can simultaneously follow the formation of individual tertiary contacts within RNA on time scales ranging from millisecond to minutes. This breadth of accessible timescales allows direct visualization of a large proportion of the tertiary transitions of Tetrahymena ribozyme folding. Synchrotron x-ray footprinting will be used in Overall Specific Aim 1 in conjunction with small angle x-ray scattering to determine if the ribozyme "collapses" nonspecifically prior to proceeding down a specific folding pathway or whether the specific tertiary contacts guide the initial electrostatic collapse. In Overall Specific Aim 2, hydroxyl radical footprinting will be used to explore the structure of the structures present in the unfolded ribozyme. Time-resolved synchrotron x-ray footprinting studies will characterize the folding pathways that predominate at physiological conditions and the native and non-native intermediates of these pathways. Selected ribozyme mutants that perturb the stability of the P4-P6 domain, the peripheral helices and the catalytic core will be analyzed by synchrotron x-ray footprinting in order to explore the structures and lifetimes of the intermediate species. Overall Specific Aim 3 seeks to map the folding 'landscape' for the Tetrahymena ribozyme and the preferred pathways followed by the RNA within it. Solution variables including monovalent and divalent ion concentration will be probed individually and in combination in order to comprehensively map the preferred pathways within the RNA folding landscape. Lastly, the folding of ribozymes in which individual tertiary contacts have been perturbed by mutation will be analyzed in order to distinguish the contributions of inter and intradomain interactions to ribozyme folding. These studies will identify the tertiary contacts that constitute kinetically trapped intermediates, identify preferred pathways through the folding landscape and generate predictions of tertiary contacts that constitute kinetically trapped intermediates for analysis by single molecule FRET.