Experimentally Guided Ribosomal RNA Modeling

描述(申请人提供)：本提案寻求继续支持通过冷冻电子显微镜和单粒子重建来探索活跃在蛋白质合成中的核糖体的结构和功能。在追求这一方法的过程中，关于这一过程的动力学已经有了几个开创性的发现；其中包括在mRNA-tRNA移位过程中的棘轮运动，以及当氨基酰-tRNA进入核糖体时的弹簧状变形。所提出的研究重点有两个：以高分辨率表征在解码和移位过程中适当稳定的过渡态，以及使用预平衡样品的时间分辨低温电子显微镜(MS范围)来研究这两个过程的时间过程。在奥尔巴尼沃兹沃斯中心的NCRR/NIH RVBC资源中心开发的新型单片式微流控混合和喷涂设备将与该资源合作进行测试和使用，该技术稍后将转移到哥伦比亚实验室。样本将从约翰·霍普金斯大学的雷切尔·格林博士和乌普萨拉大学的曼斯·埃伦伯格博士那里获得。其他合作领域包括异质样品的分类和分子动力学模拟。当密度图以足够的分辨率获得时，将通过灵活的拟合进行分析，并在丰富的结构、动力学、单分子FRET和生化数据的背景下进行解释。 与公共健康相关：核糖体在所有细胞中进行蛋白质合成。这项研究将进一步加深对其功能动力学的理解，从而有助于从根本上理解所有的生命过程。具体地说，了解细菌核糖体的功能有助于进一步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.