IIBR RoL: Applying innovative structural tools to highlight RNA's structural dynamics as RNA-protein complexes self-assemble

生命功能是由包括蛋白质和RNA在内的大生物分子的相互作用来支持的。尽管其中一些复合体的形状或结构已经被可视化，但人们对它们的组装动力学知之甚少。这些不同的组件如何相互作用，创造出功能超过任何一个单独的功能的复合体？这是一个具有挑战性的问题，因为组件的运动相互交织。通过这个项目，新的工具将被应用来强调在组装复合体过程中只有一个大分子物种的运动。虽然蛋白质和核糖核酸都存在，但只有核糖核酸是可见的。学生们将设计、建造和应用这项新技术来研究一种小型植物病毒的自组装，这种病毒是一种复杂的结构，由包裹着核酸核心的蛋白质壳组成。这些研究将使我们能够更清楚地定义管理核酸和蛋白质加入生物复合体的规则。许多重要的生物组装是由不同的大分子成分组成的，例如蛋白质和核酸。尽管它们很重要，但蛋白质数据库中只有不到2%的静态结构反映了RNA-蛋白质复合体。关于RNA和蛋白质成分之间的动态相互作用更是知之甚少。虽然人们已经花费了大量的精力来观察一种类型的大分子自组装，例如蛋白质折叠，但由于存在多个物种，解释多组分机器的动态结构信息要困难得多。无法将信号从不同的分量中分离出来，这就排除了简单的解释。该项目将开发和创新现有的实验基础设施，以创建只在一个物种的背景上突出另一个物种的信号的工具，从而极大地简化对复合体动态的测量。将构建快速结合蛋白质和RNA前体的混合器，使实验人员能够跟踪复杂组装过程中仅RNA成分的结构变化。一种小型植物病毒将作为这些研究的模型系统。两种互补技术(小角X射线散射和单分子荧光)的发展和应用将为这一自组装问题提供一个独特的视角。一旦确定，这些方法可以应用于其他RNA-蛋白质复合体，收集数据以阐明它们组装的规则。按主题排序的项目结果将发布在：https://pollack.research.engineering.cornell.edu/all_publications/.该项目由生物基础设施计划生物研究基础设施创新计划、分子和细胞生物科学部门的分子生物物理和遗传机制集群以及生物主管新兴前沿办公室的生命规则倡议共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Life functions are supported by the interaction of large biological molecules, including proteins and RNA. Although the shapes or structures of some of these complexes have been visualized, very little is known about their dynamics of assembly. How do these differing components interact to create complexes whose function exceeds that of either alone? This is a challenging problem because of the intertwined motions of the components. Through this project, new tools will be applied to highlight the motions of just one macromolecular species during the assembly of a complex. Although both proteins and RNA are present, only the RNA is visible. Students will design, build and apply the new technology to study the self-assembly of a small plant virus, an intricate structure consisting of a protein shell surrounding a nucleic acid core. These studies will allow us to more clearly define the rules that govern the joining of nucleic acids and proteins into biological complexes. Many important biological assembles are comprised of distinct macromolecular components, such as proteins in conjunction with the nucleic acids. Despite their importance, less than 2% of the static structures in the protein data bank reflect RNA-protein complexes. Even less is known about the dynamic interactions between RNA and protein components. Although much effort has been expended in watching one type macromolecule self-assemble, e.g. protein folding, it is much more difficult to interpret dynamic structural information for multicomponent machines because multiple species are present. The inability to separate the signal from the distinct components precludes a simple interpretation. This project will exploit and innovate existing experimental infrastructure to create tools that highlight the signal of only one species on the background of the other, vastly simplifying measurements of complexes' dynamics. Mixers that rapidly combine the protein and RNA precursors will be constructed and will allow experimenters to track the changing structures of only the RNA component during complex assembly. A small plant virus will serve as a model system for these studies. The development and application of two complementary techniques (small angle x-ray scattering and single molecule fluorescence) will offer a unique perspective on this self-assembly problem. Once established, these methods can be applied other RNA-protein complexes, gathering data to elucidate the rules governing their assembly. Project results, sorted by topic, will be posted at: https://pollack.research.engineering.cornell.edu/all_publications/. This project is jointly funded by the Division of Biological Infrastructure program for Infrastructure Innovation for Biological Research, the Molecular Biophysics and Genetic Mechanism Clusters in the Division of Molecular and Cellular Biosciences, and the Rules of Life initiative in the Emerging Frontiers office of the Biology Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.