Long Range Interactions in Mu and Bacterial DNA

描述（由申请人提供）：染色体动力学和体内引导DNA运动的蛋白质是细胞复制、基因表达、遗传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，限制超螺旋扩散。未来四年我们研究的主要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螺旋运动的机械机制。我们有具体的目标。1)将促旋酶和细菌凝聚素MukB的活性与类核致密化联系起来。这一目标包括一个新的组成部分，涉及DNA旋转酶生物化学和遗传方法，从E。大肠杆菌/沙门氏菌属比较。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。控制染色体结构片段的候选“结构域”或蛋白质将通过4个测试的挑战来测量结构域行为。这些测试包括分析特定基因在8个不同位置改变超螺旋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。将结构域和DNA运动连接到结构。我们将测试结构域的环模型，并定义高度转录的核糖体RNA操纵子的动态特性。当细胞在丰富的培养基中快速生长时，所有RNA合成的70%用于稳定的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的基因组中的位置。我们还将确定野生型细菌中的转录是否能产生“正超螺旋波”。“2）将域和运动连接到结构。我们将测试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成环的假设。3)使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-operator模块的结构域。研究将确定模块如何在体内表现为染色体动力学元素时，未被占用，当装饰有不同水平的荧光结合蛋白，以及如何诱导物改变超螺旋动力学与结合阻遏物的网站。在这些实验的过程中，我们将荧光蛋白结合模块放入E。利用上一个资助期开发的有效重组方法，在20个不同位置上对大肠杆菌和沙门氏菌染色体进行了重组，并确定了当染色体片段通过位点特异性重组从主体分离时发生了什么。公共卫生相关性：该项目旨在开发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必需蛋白质也存在于真核生物直到人类中。这项工作为开发新的抗生素以对抗病原微生物和解决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.