Long Range Interactions in Mu and Bacterial DNA

描述（由申请人提供）：染色体动力学和在体内引导DNA运动的蛋白质是细胞复制、基因表达、基因重组和达尔文进化的关键决定因素。最近的研究表明，细菌染色体被组织成大约400个独立的结构域，这些结构域限制了超卷曲的扩散。未来四年，我们的主要研究重点将是确定关键蛋白质，并剖析控制细菌染色体结构和活细胞内超级线圈运动的机械机制。我们有明确的目标。1)将回转酶和细菌凝聚蛋白MukB的活性与类核压实联系起来。这一目标包括一个新的组成部分，涉及DNA旋切酶生物化学和遗传方法，从大肠杆菌/沙门氏菌物种比较中进化而来。遗传选择将用于鉴定在400结构域染色体中形成随机和序列特异性结构域边界的蛋白质。候选“结构域蛋白”或控制染色体结构片段的蛋白质将通过4项测试来测量结构域行为。这些测试包括分析特定基因在8个不同位置改变超线圈密度和位点特异性分辨率效率的能力，测试它们对核糖体RNA操纵子的影响，以及测量每个基因的平均结构域大小。连接域和DNA运动到结构。我们将测试区域结构的环模型，并定义高度转录的核糖体RNA操纵子的动态特性。当细胞在富培养基中快速生长时，70%的RNA合成都是稳定的RNA（核糖体和tRNA基因）。新的实验将测试这些区域是否形成特定的转录环，并确定高度转录的基因在折叠基因组中的位置。我们还将确定WT细菌的转录是否能产生“正超线圈波”。2)将领域和运动与结构联系起来。我们将测试一个用于组织高度转录的蛋白质编码基因、核糖体RNA操纵子和tRNA操纵子的环模型。我们还将利用染色体构象捕获技术来证明我们关于环的假设。3)利用荧光细胞技术将平均染色体结构与单细胞行为联系起来。将分析作为染色体行为的细胞生物学标记的Lac-和tet -算子模块的结构域结构。研究将确定模块在体内未被占用时如何表现为染色体动力学元件，当被不同水平的荧光结合蛋白修饰时，以及诱导剂如何改变具有结合阻遏物的位点的超线圈动力学。在这些实验过程中，我们将利用上次拨款期开发的高效重组方法，在大肠杆菌和沙门氏菌染色体的20个不同位置放置荧光蛋白结合模块，并确定通过位点特异性重组将染色体片段与主体分离时会发生什么。公共卫生相关性：本项目旨在建立染色体组织的分子模型，并测量活细胞内的DNA动力学。许多参与细菌细胞分裂的基本蛋白质也在真核生物中被发现，直到人类。这项工作为开发新的抗生素来对抗病原微生物和解决关于染色体在细胞生长过程中如何解开的老问题提供了理论基础。

DESCRIPTION (provided by applicant): Chromosome dynamics and the proteins that channel DNA movement in vivo are critical determinants of cell replication, gene expression, genetic recombination, and Darwinian evolution. Recent studies have demonstrated that bacterial chromosomes are organized into about 400 independent domains that limit supercoiling diffusion. The primary focus of our research over the next four years will be to identify the critical proteins and dissect the mechanical mechanisms that control bacterial chromosome structure and supercoil movement inside living cells. We have specific aims. 1) Connect the activities of gyrase and the bacterial condensin, MukB, to nucleoid compaction. This aim includes a new component involving DNA gyrase biochemistry and genetic methods that evolved from the E. coli/Salmonella species comparison. Genetic selections will be used to identify the proteins that form both the stochastic and sequence-specific domain boundaries in the 400 domain chromosome. Candidate "Domainins" or proteins that control a segment of chromosome structure will be run through a gauntlet of 4 tests to measure domain behavior. These tests include analyzing specific genes for their ability to change supercoil density and site-specific resolution efficiency at eight different locations, testing their effect on ribosomal RNA operons, and measuring the average domain size for each gene. Connect domains and DNA movement to structure. We will test a loop model for domain structure and define the dynamic characteristics of highly transcribed ribosomal RNA operons. As cells grow rapidly in rich media, 70% of all RNA synthesis is devoted to stable RNAs (ribosomal and tRNA genes). New experiments will test whether these regions form specific transcription loops and determine where the highly transcribed genes are in the folded genome. We will also establish whether or not transcription in WT bacteria can generate "waves of positive supercoils." 2) Connect domains and movement to structure. We will test a loop model for organizing highly transcribed protein-encoding genes, ribosomal RNA operons, and tRNA operons. We will also exploit the chromosome conformation capture technology to prove our hypothesis on looping. 3) Connect the average chromosome structure to single cell behavior using fluorescent cell technology. The domain structure of Lac- and Tet-operator modules that serve as cell biological markers of chromosome behavior will be analyzed. Studies will determine how modules behave as chromosome dynamics elements in vivo when unoccupied, when decorated with different levels of fluorescent binding protein, and how inducer changes supercoil dynamics for sites with bound repressors. In the course of these experiments, we will place fluorescent protein binding modules into the E. coli and Salmonella chromosome at 20 different positions using efficient recombination methods developed in the last grant period, and determine what happens when a segment of chromosome is separated from the main body by site specific recombination. PUBLIC HEALTH RELEVANCE: This project aims to develop a molecular model of chromosome organization and measure DNA dynamics inside living cells. Many essential proteins that participate in bacterial cell division are also found in eukaryotic organisms up to humans. This work provides a rationale for developing new antibiotics to fight pathogenic microorganisms and to solve old problems about how chromosomes become disentangled during cell growth.