Time-resolved hydroxyl radical footprinting

时间分辨同步加速器x射线羟基自由基足迹可以在毫秒到分钟的时间尺度上同时跟踪RNA内单个三级接触的形成。这种可访问的时间尺度的宽度允许直接可视化四膜虫核酶折叠的三级跃迁的很大比例。同步加速器x射线足迹将与小角度x射线散射一起用于总体特异性目标1，以确定核酶是否在沿着特定折叠途径进行之前非特异性“崩溃”，或者特定的三级接触是否引导初始静电崩溃。在总体特异性目标2中，羟基自由基足迹将用于探索未折叠核酶中存在的结构的结构。时间分辨同步加速器x射线足迹研究将描述在生理条件下占主导地位的折叠途径以及这些途径的原生和非原生中间体。选定的干扰P4-P6结构域、外围螺旋和催化核心稳定性的核酶突变体将通过同步加速器x射线足迹分析，以探索中间物种的结构和寿命。总体特异性目标3旨在绘制四膜核糖酶的折叠“景观”，以及其中的RNA遵循的首选途径。包括单价和二价离子浓度在内的溶液变量将被单独和组合探测，以便全面绘制RNA折叠景观中的首选途径。最后，将分析单个三级接触受到突变干扰的核酶折叠，以区分结构域间和结构域内相互作用对蛋白质的贡献

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.