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.

项目概要 该应用描述了两个重要方面的广泛生物物理特征 细菌细胞生理学。首先，整个大肠杆菌蛋白质组的动态将被量化 作为生长速率和营养限制的函数，包括蛋白质周转率的测量。 这组细菌蛋白质经过精心控制，以优化细菌的适应性和生长。 各种条件下的细菌。蛋白质的量专门用于各种 生物合成途径以明确的方式变化，可以简单地建模为经济 分配问题。其次，将使用以下方法研究核糖体组装过程 电子显微镜、质谱、单分子荧光和光学显微镜。 在扰动条件下积累的中间体的电子显微镜揭示了 可以排序到假定的组装路径中的结构的分布。单分子 荧光可以监测复杂组装过程中的各个步骤。 最后，我们将体外研究扩展到研究基因座的超微结构。 结合光学显微镜和冷冻电子技术在细胞中组装核糖体 断层扫描。总体目标是开发一个完整的机械框架来组装 最大的细胞机器，负责所有蛋白质的合成，并且需要 几乎是快速分裂细胞能量预算的三分之一。