Assembly of multifunctional domains at bacterial cell poles

描述(由申请人提供)：这项工作的目标是了解管理蛋白质组装成有组织的细胞域的分子机制。这些实验将检查细菌细胞中的结构域组装，但结构域的建立是所有细胞类型中都会发生的普遍现象。考虑到这一项目作为原则的证明，同样的实验方法可以转移到真核系统，并用于在人类生物学中做出新的发现。这项工作将集中在棒状新月弯杆菌中的细胞极组装上，这将在连续细胞质的相反两端创建两个功能不同的极点。这些极点是复杂的结构域，执行多种功能，包括鞭毛组装、染色体定位和转录因子活性的调节。将在两个遗传屏幕中发现极性组装因子：一个是视觉屏幕，直接识别已知极性蛋白质正确定位所需的基因，另一个是新的极性蛋白质的生物信息屏幕。对于在筛选中发现的每个基因，组装过程中的上游和下游因素将使用遗传、显微和生物化学方法进行鉴定。生物信息学筛选已经产生了一种新的蛋白质，它似乎使用染色体复制作为在细胞极点组装的线索。分析的一个关键方面将是使用冷冻电子显微镜断层扫描的蛋白质特定标记技术精确确定蛋白质定位。这将揭示蛋白质和功能复合体在结构域超微结构背景下的排列，从而表征遗传和生物化学方法无法获得的组装方面。该项目将定义组装步骤的时间顺序，并显示单个组件如何放置在电池杆的整体架构中，提供该领域结构的详细四维视图。蛋白质是作为单独的亚基组成的，必须找到一种方法与其他蛋白质结合在一起，形成具有生物功能的更大的复合体。这种组装必须正确地发生，才能使我们的细胞生长和分裂，而问题的另一面是，我们可能会在病毒和细菌中阻止这一过程，以抑制它们的生长。我正在研究细菌中的蛋白质组装，这可能直接导致新抗生素的开发。这项工作还将对我们关于复杂的组装如何有助于所有生命形式的细胞功能的知识产生更广泛的影响。

DESCRIPTION (provided by applicant): The goal of this work is to understand the molecular mechanisms that govern the assembly of proteins into an organized cellular domain. The experiments will examine domain assembly in bacterial cells, but the establishment of domains is a general phenomenon that occurs in all cell types. Given this project as proof- of-principle, the same experimental approach could be transferred to eukaryotic systems and used to make new discoveries in human biology. The work will focus on cell pole assembly in the rod shaped Caulobacter crescentus, which creates two functionally distinct poles at opposite ends of a contiguous cytoplasm. These poles are complex domains that carry out multiple functions, including flagellar assembly, chromosome positioning, and the regulation of transcription factor activity. Polar assembly factors will be discovered in two genetic screens: one a visual screen to directly identify genes that are required for the proper localization of known polar proteins, the other a bioinformatic screen for new polar proteins. For every gene identified in the screens, upstream and downstream factors in the assembly process will be identified using genetic, microscopic, and biochemical methods. The bioinformatic screen has already yielded a novel protein that seems to use chromosome replication as a cue for assembly at the cell pole. A key aspect of the analysis will be a precise determination of protein localization using protein-specific labeling techniques for cryo-EM tomography. This will reveal the arrangement of proteins and functional complexes in the context of domain ultrastructure, thereby characterizing an aspect of assembly that is inaccessible by genetic and biochemical methods. The project will define a temporal order of assembly steps and show how individual components are placed in the overall architecture of the cell pole, providing a detailed four dimensional view of the construction of this domain. Proteins are made as individual subunits, and must find a way to fit together with other proteins to create larger complexes with biological function. Such assembly must occur properly in order for our cells to grow and divide, and the flip side of the coin is that we may block this process in viruses and bacteria to inhibit their growth. I am studying protein assembly in a bacterium, which could lead directly to the development of new antibiotics. The work will also have broader impact on our knowledge of how complex assembly contributes to cellular function in all life forms.