Dynamics of the Bacterial Ribosome and Proteome

项目摘要 本申请描述了两个重要方面的广泛基础的生物物理表征 细菌细胞生理学。首先，对整个E.将对大肠杆菌蛋白质组进行定量 作为生长速率和营养限制的函数，包括蛋白质周转的测量。 这组细菌蛋白被仔细控制以优化细菌的适应性和生长。 在各种条件下的细菌。蛋白质的量用于各种 生物合成途径以确定的方式变化，可以简单地建模为经济 分配问题。第二，核糖体组装的过程将使用 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显示， 结构的分布，可以被安排到一个假定的组装途径。单分子 荧光允许监测复杂组装过程中的各个步骤。 最后，我们将扩大体外研究，研究基因座的超微结构， 核糖体组装在细胞中，使用光学显微镜和冷冻电子相结合 断层扫描总体目标是开发一个完整的机械框架， 最大的细胞机器，负责所有蛋白质的合成， 几乎是快速分裂细胞能量预算的三分之一。

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.