Experimentally Guided Ribosomal RNA Modeling

描述（由申请人提供）：本提案寻求通过低温电子显微镜和单粒子重建继续支持探索积极参与蛋白质合成的核糖体的结构和功能。在追求这种方法的过程中，关于这一过程的动力学已经有了几个开创性的发现；其中包括mRNA-tRNA易位时的棘轮运动和氨基基trna进入核糖体时的弹簧样变形。所提出的研究的重点是双重的：以高分辨率表征解码和易位过程中适当稳定的过渡状态，并使用前平衡样品的时间分辨低温电子显微镜（ms范围）研究这两个过程的时间过程。位于奥尔巴尼的沃兹沃斯中心的NCRR/NIH RVBC资源将与该资源合作测试和使用一种新型的单片微流体混合和喷涂装置，该技术随后将转移到哥伦比亚实验室。样本将从合作者dr。约翰霍普金斯大学的Rachel Green和乌普萨拉大学的Mans Ehrenberg。其他的合作是在异质样品的分类和分子动力学模拟领域。当以足够的分辨率获得密度图时，将通过灵活的拟合进行分析，并在结构，动力学，单分子FRET和生化数据的丰富背景下进行解释。

DESCRIPTION (provided by applicant): This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy and single-particle reconstruction. Several seminal discoveries regarding the dynamics of this process have been made in pursuit of this approach; among these the ratchet motion during mRNA-tRNA translocation and the spring-like deformation of the aminoacyl-tRNA as it enters the ribosome. The emphasis of the proposed studies is twofold: to characterize suitably stabilized transition states during both decoding and translocation at high resolution, and to study the time course of both processes using time-resolved cryo-electron microscopy (ms range) of pre-equilibrium samples. A novel monolithic microfluidic mixing and spraying device developed at the NCRR/NIH RVBC Resource at the Wadsworth Center in Albany will be tested and used in collaboration with the Resource, and the technology will be later transferred to the Columbia lab. Samples will be obtained from collaborators Drs. Rachel Green, Johns Hopkins University and Mans Ehrenberg, Uppsala University. Additional collaborations are in the areas of classification of heterogeneous samples and Molecular Dynamics simulations. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of structural, kinetics, single-molecule FRET, and biochemical data. PUBLIC HEALTH RELEVANCE: The ribosome performs protein synthesis in all cells. The research proposed will further the understanding of its functional dynamics, and thus contribute to a fundamental understanding of all life processes. Specifically, understanding the function of bacterial ribosomes is furthering our ability to combat drug resistance.