Dynamics of the Bacterial Ribosome and Proteome

项目总结 本申请描述了两个重要方面的广泛生物物理表征 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 作为生长速度和营养限制的函数，包括蛋白质周转的测量。 细菌蛋白质组受到精心控制，以优化细菌的适合性和生长 细菌在各种不同的条件下。不同种类蛋白质的含量 生物合成途径以一种定义的方式变化，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用 电子显微镜、质谱学、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 可以排列成假定的组装路径的结构的分布。单分子 荧光可以监测复杂组装过程中的各个步骤。 最后，我们将扩大体外研究，以研究基因座的超微结构。 使用光学显微镜和低温电子相结合的方法在细胞中组装核糖体 体层摄影术。总体目标是开发一个完整的机械框架，用于组装 最大的细胞机器，负责所有蛋白质的合成，这需要 几乎是一个快速分裂细胞的能源预算的三分之一。

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.