Dynamics of the Bacterial Ribosome and Proteome

细菌核糖体和蛋白质组的动力学

• 批准号: 10380121
• 负责人: James R Williamson
• 金额: $ 69.98万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10380121
• 关键词: Applications Grants; Bacteria; Bacterial Proteins; Budgets; Cell physiology; Cells; Complex; Cryo-electron tomography; Economics; Electron Microscopy; Escherichia coli; Fluorescence; Fluorescence Microscopy; Goals; Growth; In Vitro; Individual; Mass Spectrum Analysis; Measurement; Modeling; Monitor; Nutrient; Pathway interactions; Process; Protein Biosynthesis; Proteins; Proteome; Ribosomes; Structure; base; biophysical properties; fitness; light microscopy; protein degradation; response; single molecule

PROJECT SUMMARY This application describes broadly based biophysical characterization of two important aspects of bacterial cell physiology. First, the dynamics of the entire E. coli proteome will be quantified as a function of growth rate and nutrient limitation, including measurement of protein turnover. The set of bacterial proteins are carefully controlled to optimize the fitness and growth of bacteria under a wide variety of conditions. The amount of protein devoted to various biosynthetic pathway varies in a defined way that can be simply modeled as an economic allocation problem. Second, the process of ribosome assembly will be investigated using electron microscopy, mass spectrometry, single molecule fluorescence, and light microscopy. Electron microscopy of intermediates that accumulate under perturbed conditions reveals a distribution of structures that can be ordered into a putative assembly pathway. Single molecule fluorescence allows the monitoring of individual steps in the complex assembly process. Finally, we will extend the in vitro studies toward studying the ultrastructure of the locus of ribosome assembly in cells, using a combination of light microscopy and cryo-electron tomography. The overall goal is to develop a complete mechanistic framework for assembly of the largest cellular machine, that is responsible for all protein synthesis, and that requires almost a third of the energy budget for a rapidly dividing cell.

项目摘要 该应用描述了两个重要方面的广泛基于生物物理的表征 细菌细胞生理。首先，将量化整个大肠杆菌蛋白质组的动力学 作为生长速率和养分限制的函数，包括蛋白质周转的测量。 仔细控制了一组细菌蛋白，以优化 细菌在各种条件下。致力于各种的蛋白质量 生物合成途径以一种可以简单地建模为经济的定义方式变化 分配问题。其次，将使用核糖体组装过程 电子显微镜，质谱，单分子荧光和光显微镜。 在扰动条件下积累的中间体的电子显微镜表明 可以订购到推定的组装途径的结构的分布。单分子 荧光允许监视复杂组装过程中的各个步骤。 最后，我们将将体外研究扩展到研究的超微结构 核糖体组装在细胞中，使用光学显微镜和冷冻电子的组合 断层扫描。总体目标是开发一个完整的机械框架以组装 最大的蜂窝机，负责所有蛋白质合成，这需要 快速分裂的单元格的能源预算的几乎三分之一。