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几分钟的时间尺度上同时跟踪RNA中单个三级接触的形成。这种可获得的时间尺度的广度允许直接看到四膜虫核酶折叠的很大比例的第三次转变。同步加速器x射线足迹将与小角x射线散射一起用于整个特定的目标1，以确定核酶是否在沿着特定的折叠路径进行之前非特定地“塌陷”，或者特定的三级接触是否引导初始的静电塌陷。在整个特定目标2中，将使用羟基自由基足迹来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射线足迹研究将表征在生理条件下占主导地位的折叠途径以及这些途径的天然和非天然中间体。选择扰乱P4-P6结构域、外周螺旋和催化核心稳定性的核酶突变体，将用同步加速器x射线足迹法进行分析，以探索中间物种的结构和寿命。总体上，具体目标3试图绘制四膜虫核酶的折叠‘景观’以及其中的RNA遵循的首选路径。包括一价离子浓度和二价离子浓度在内的溶液变量将单独和结合进行探测，以全面绘制RNA折叠景观中的首选路径。最后，将分析核酶的折叠，在这些核酶中，个体的三级接触受到突变的干扰，以区分结构域间和结构域内相互作用对 核酶折叠。这些研究将确定构成动力学捕获中间体的三级接触，确定通过折叠格局的首选路径，并生成构成动力学捕获中间体的三级接触的预测，以供单分子FRET分析。

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.