Experimentally Guided Ribosomal RNA Modeling

描述（由申请人提供）：本提案寻求通过冷冻电子显微镜和单粒子重建对积极参与蛋白质合成的核糖体的结构和功能进行探索的持续支持。在追求这种方法的过程中，已经取得了一些关于这个过程的动力学的开创性发现;其中包括mRNA-tRNA易位过程中的棘轮运动和氨酰-tRNA进入核糖体时的弹簧状变形。建议的研究的重点是双重的：适当稳定的过渡态的特点在解码和易位在高分辨率，并研究这两个过程的时间过程中使用的时间分辨低温电子显微镜（ms范围）的预平衡样品。在奥尔巴尼的沃兹沃斯中心的NCRR/NIH RVBC资源开发的一种新型单片微流体混合和喷雾装置将与该资源合作进行测试和使用，该技术随后将转移到哥伦比亚实验室。样本将从约翰霍普金斯大学的Rachel绿色博士和乌普萨拉大学的Mans Ehrberg博士处获得。其他合作是在异质样品的分类和分子动力学模拟领域。当在足够的分辨率下获得密度图时，将通过灵活的拟合进行分析，并在结构、动力学、单分子FRET和生物化学数据的丰富背景下进行解释。 公共卫生相关性：核糖体在所有细胞中进行蛋白质合成。这项研究将进一步了解其功能动力学，从而有助于对所有生命过程的基本理解。具体来说，了解细菌核糖体的功能有助于提高我们对抗耐药性的能力。

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.