Time-resolved small angle X-ray scattering

RNA在许多生物过程中起着重要作用，但其构象动力学尚未在最基本的水平上被理解：自组装或折叠。本计划的长期目标是完整表征大RNA的折叠：四膜虫核酶。这一建议提出了一系列的研究，旨在探测这种核酶的全球构象，因为它通过一个初始的快速崩溃，并随后显着的压缩步骤进行折叠。使用一个演示的组合的流动池，设计用于同步小角度X射线散射，允许访问的时间尺度上的结构信息，从亚毫秒到几分钟后开始折叠。 以前的工作表明，在整个政权的散射信号的显着变化，对应于大规模的结构rearragements与压实和折叠。 为实现我们的长期目标，提出了三个具体目标。为了实现第一个，时间分辨小角度X射线散射将用于表征在折叠开始时发生的快速塌陷。速率常数和瞬态结构信息将在不同的解决方案和序列条件下获得调查的作用，抗衡离子崩溃和测试结构模型的紧凑状态。第二个具体目标是针对后来的中间体，包括部分折叠状态的结构表征，使用小角度X射线散射。展开的反向过程也将被研究。最后的具体目标探测全局构象沿着的首选折叠途径，在一价和二价阳离子的存在下填充。将在此目标下探索更接近生理学的溶液条件。折叠将从离散的未折叠状态组开始，以确定沿沿着单独路径的瞬态特性。来自这些小角散射实验的全局结构信息将与从其他项目成员获得的详细局部信息相结合，以实现对该分子折叠的尽可能深入的了解。

RNA plays an important role in many biological processes, but its conformational dynamics are not yet understood on the most fundamental level: self-assembly or folding. The long term objective of this Program Project is the complete characterization of folding of a large RNA: the Tetrahymena ribozyme. This proposal presents aseries of studies designed to probe the global conformation of this ribozyme as it proceeds towards folding through an initial rapid collapse, and subsequent significant compaction steps. The use of a demonstrated combination of flow cells, designed for synchrotron small angle x-ray scattering, allows access to structural information on time scales ranging from sub-millisecond to minutes after the initiation of folding. Previous work shows significant changes in the scattering signals throughout this regime, corresponding to large scale structural rearragements associated with compaction and folding. Three specific aims are presented to achieve our long term goal. To realize the first, time resolved small angle x-ray scattering will be used to characterize the rapid collapse that occurs at the onset of folding. Rate constants and transient structural information will be obtained under different solution and sequence conditions to investigate the role of counterions in collapse and to test structural models of compact states. The second specific aim is directed at structural characterization of later intermediates including partially folded states, using small angle x-ray scattering. The reverse process of unfolding will also be studied. The final specific aim probes the global conformations along the preferred folding pathways that are populated in the presence of both monovalent and divalent cations. Solution conditions that more closely resemble physiological will be explored under this aim. Folding will be initiated from discrete sets of unfolded states to determine the characteristics of transients along separate pathways. The global structural information from these small angle scattering experiments will be integrated with detailed, local information acquired from other Project members to achieve the deepest possible understanding of the folding of this molecule.