Molecular Dissection of Chromosome Folding in E coli

描述：(申请人提供)：本建议书涉及 染色体所需基因和蛋白质的分子分析 原核细胞中的凝聚。该项目的最终目标是 彻底了解这种复杂的、基本的分子机制 进程。大肠杆菌被用作这些研究的模型系统，因为 广泛的遗传和生化工具，随时可供实验使用。 该提案的目的是研究新的和关键的细胞成分 它们是将染色体折叠成紧凑而又有功能的 结构。这些研究中确定的组成部分和机制将导致 以更好地理解这一重要过程是如何完成的。vt.给出 染色体折叠对细胞活力至关重要，很可能 这些研究将为治疗剂确定潜在的新靶点。 利用抗DNA去折叠剂的新型遗传选择 樟脑，一种以前未知的冷凝系统，包含三种成分， CRCA。CSPE和CrcB。该系统的核心组件是 CSPE是一种69个氨基酸的核酸结合蛋白。所描述的实验 在本提案中，进行了完整的结构和功能分析 CSPE.从基因上分离不同表型的CSPE的突变将 使用两种独立的方法进行隔离。初步研究表明，其中一个 在这些方法中，可以成功地使用。这些蛋白质来自于 野生型基因和突变基因将被分离并鉴定为 一系列体外试验。 原核细胞含有几种小的DNA结合蛋白，这些蛋白在 在染色体折叠中。所有这些蛋白质都参与了许多不同的 细胞过程。在这方面，CSPE是这个班级的另一个成员。 是什么使CSPE有别于其他蛋白质，并使其最具顺应性 研究染色体折叠的直接作用的班级成员是 通过突变从基因上分离其不同功能的能力。这个 对本提案中描述的突变CSPE蛋白的表征将 让我们直接了解这类小分子如何与核酸结合 蛋白质在染色体折叠的基本过程中发挥作用。

DESCRIPTION: (provided by applicant): This proposal is concerned with the molecular analysis of the genes and proteins required for chromosome condensation in prokaryotic cells. The ultimate goal of the project is to thoroughly understand the molecular mechanisms of this complex, fundamental process. E. coli is used as the model system for these studies due to the extensive genetic and biochemical tools readily available for experimental use. The intent of the proposal is to study novel and critical cellular components that are required to fold the chromosome into a compact yet functional structure. The components and mechanisms identified in these studies will lead to a greater understanding of how this essential process is accomplished. Given that chromosome folding is essential for cell viability, it is likely that these studies will identify potential new targets for therapeutic agents. Using the novel genetic selection of resistance to the DNA unfolding agent camphor, a previously unknown condensing system containing three components, CrcA. CspE and CrcB, was identified. The central component of this system is CspE, a 69 amino acid, nucleic acid-binding protein. The experiments described in this proposal undertake a complete structural and functional analysis of CspE. Mutations that genetically separate the different phenotypes of cspE will be isolated using two, independent approaches. Pilot studies indicate that one of these approaches can be used successfully. The proteins produced from the wild-type gene and the mutated genes will be isolated and characterized in a series of in vitro assays. Prokaryotic cells contain several small, DNA-binding proteins that play a role in chromosome folding. All of these proteins are involved in many, different cellular processes. In this respect, CspE is yet another member of the class. What sets CspE apart from these others proteins and makes it the most amenable member of the class for the study of a direct role in chromosome folding is the ability to genetically separate its different functions by mutation. The characterization of the mutant CspE proteins described in this proposal will lead to a direct understanding of how this class of small, nucleic acid-binding proteins function in the essential process of chromosome folding.