Regulation of Gene Transcription

A 部分：染色体结构和功能。根据我们对基因调控机制的研究，我们之前提出细菌染色体（核）具有决定基因表达的条件依赖的确定结构。 HU 是真细菌中最保守的核相关蛋白，但人们对它如何影响整体染色体组织和基因表达知之甚少。 (i) 使用单分子追踪，我们证明 HU 与染色体 DNA 表现出非特异性、弱且短暂的相互作用。这些相互作用主要由三个保守的、表面暴露的赖氨酸残基 (triK) 介导，此前已证明这些残基负责与 DNA 的非特异性结合。 HUa(triKA) 突变体中这些微弱、短暂的相互作用的丧失会导致核仁过度浓缩和错误分离。与 HUa(triKA) 突变体相比，突变 HUa 亚基中的保守脯氨酸残基 (P63A)、删除 HUb 亚基或删除与类核相关的 naRNA（之前均涉及 HU 与扭结或十字形 DNA 的高亲和力结合），导致 HU 的相互作用动力学变化不太显着，但类核高度扩展。我们的结果表明，HU 通过与染色体 DNA 的不同相互作用，在维持适当的核体积方面发挥着双重作用。一方面，HU 通过特定的 DNA 结构结合相互作用压缩类核。另一方面，它通过与大量染色体的许多非特异性、弱的和短暂的相互作用来解压缩类核。这种动态相互作用可能有助于细菌核的粘弹性和流动性，以促进适当的染色体功能。 (ii) 通过软 X 射线断层扫描对近天然、未标记的大肠杆菌细胞进行成像，我们发现 HU 通过促进形成被较少浓缩的孤立域包围的致密浓缩核心来重塑核仁。细胞生长和环境适应过程中的类核重塑与 pH 和离子强度控制的分子开关相关，分子开关调节 HUaa 依赖性分子间 DNA 捆绑。通过晶体学和基于溶液的研究，我们表明这些效应在机制上依赖于 HUaa 混杂性形成多个静电驱动的多聚化界面。 DNA 捆绑的变化可能会通过限制 DNA 超螺旋而影响全局基因表达。总而言之，我们的研究结果揭示了 HU-DNA 相互作用在类核重塑中的关键功能，该功能可能作为转录调控的一般微生物机制，以同步细胞周期中的遗传反应并适应不断变化的环境。 (iii)我们努力阐明细菌细胞中染色体的三维结构是如何组织和维持的。使用荧光显微镜技术，我们通过直接观察活细胞内专门标记的 DNA 位点的位置来探测大肠杆菌染色体的组织。使用两个正交 ParB - parS 系统，我们能够在同一大肠杆菌菌株中同时用两种颜色标记两个 DNA 位点。我们的标记策略有一个固定基因座作为所有菌株的控制点，另外还有一个“移动”基因座，以粗粒图谱绘制整个染色体。我们的实验数据初步表明，DNA 位点之间的线性（遗传）距离间隔与其空间间隔之间存在相关性。最终，在计算模型的帮助下，我们希望通过对活细胞中不同 DNA 位点之间的大量仔细的距离测量来模拟染色体的三维组织。 (iv) 我们还证明了大肠杆菌中组蛋白样蛋白 HU 的特异性和非特异性 DNA 结合的不同生理作用。手稿正在准备中。在细菌生理学中，从染色体结构的维持到基因转录的调节。 HU 在许多病原体中都是必需的，这使其成为开发抗微生物疗法的有吸引力的目标。对 HU DNA 结合及其生理过程调节的机制理解将有助于小分子 HU 抑制剂的设计和开发。我们使用大肠杆菌作为模式生物来研究 HU 如何与染色体 DNA 相互作用并调节各种生理过程。在大肠杆菌中，HU 通过两种方式与 DNA 结合：(i) 通过三个表面暴露的赖氨酸残基（K3、K18 和 K83）与 DNA 磷酸盐形成离子键，对任何 DNA（非特异性）具有低亲和力； (ii) 通过保守的脯氨酸残基 (P63) 对包含一对扭结（结构特异性）的给定结构的扭曲 DNA 具有高亲和力，通过诱导和/或稳定扭结来介导特异性结合。我们最近证明，HU 主要通过赖氨酸残基介导的非特异性结合，以快速缔合/解离动力学与染色体 DNA 相互作用。这提供了证据表明 HU 与染色体的整体关联是通过非特异性结合实现的。顺便说一句，HU 在许多病原体中都是必需的，这使其成为开发抗微生物药物的目标。对 HU DNA 结合机制的了解将有助于 HU 抑制剂的设计和开发。 B 部分：噬菌体 Lambda 和 Gal 操纵子中的基因调控：今年我们在噬菌体 Lambda 方面的工作取得了更多进展。 RNA 聚合酶和 CI 阻遏物相互作用的研究。基因调控生物学中最容易理解的系统之一是所谓的“基因开关”。这决定了噬菌体编码的 CI 阻遏蛋白通过以定义的模式协同结合两个三方操纵子 OL（OL1、OL2 和 OL3）和 OR（OR1、OR2 和 OR3）而做出的选择。两个裂解启动子 PL 和 PR 的转录被阻断，而溶原启动子 PRM 的转录分别在低 CI 和高 CI 浓度下被激活和抑制。 PRM 的自动调节依赖于 RNA 聚合酶 (RNAP) 与 PRM 启动子的结合以及 CI 与 OR2 的结合的相互作用。通过使用纯化的体外转录系统，我们通过 DNA 和蛋白质突变分析了 PRM 处的 RNAP 和 OR2 处的 CI 之间的激活复合物。我们在 OR2 和 OR3 之间插入 5-bp 或删除 1-bp DNA，以改变 RNAP 和 CI 之间的角度方向和距离。我们还突变了 CI 的 E34K，它在 PRM 激活过程中与 RNAP 相互作用。我们得到了意想不到的发现。首先，PRM -34A 的 1-bp DNA 缺失导致在与抑制 PL 和 PR 相同的 CI 浓度下对 PRM 进行抑制。这种抑制取决于 DNA 循环以及 CI 与 OR2 的结合。其次，在 PRM 启动子位点和 OR2 位点之间插入 5 bp DNA，导致在与抑制 PL 和 PR 相同的 CI 浓度下对 PRM 进行抑制。第三，参与激活复合物的 CI 的 E34K 突变导致在相同 CI 浓度下对 PRM 的抑制，从而抑制 PL 和 PR。最后，DNA 成环增强了 PRM 的激活和抑制。结论：通过突变 CI 或插入或删除碱基对来改变 RNAP 和 CI 之间的角度方向和距离，破坏 PRM 处的 RNAP 和 OR2 处 CI 之间的激活复合物，导致 PRM 的抑制。这些意想不到的结果表明，RNAP 可能正在 OR2 处与 CI 产生负接触，从而阻止 RNAP 逃逸并抑制 PRM。未来的研究正在进行中，以了解这些变化如何导致 PRM 抑制的分子机制。目前正在进行的一项尝试是用野生型和不同突变体DNA/蛋白质模拟Prm-Ci-RNA聚合酶三元复合物的结构，并检查CI和RNA聚合酶之间“负”接触的可行性。

Part A. Chromosome structure and function. From our studies of mechanisms of gene regulation, we have previously proposed that the bacterial chromosome (nucleoid) has a condition dependent defined structure that dictates gene expression. HU is the most conserved nucleoid-associated protein in eubacteria, but how it impacts global chromosome organization and gene expression is poorly understood. (i) Using single-molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface-exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUa(triKA) mutant results in an over-condensed and mis-segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUa subunit, deleting the HUb subunit, or deleting nucleoid-associated naRNAs, each previously implicated in HU's high-affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUa(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure-binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions. (ii) By imaging of near-native, unlabeled E. coli cells by soft X-ray tomography, we showed that HU remodels nucleoids by promoting the formation of a dense condensed core surrounded by less condensed isolated domains. Nucleoid remodeling during cell growth and environmental adaptation correlate with pH and ionic strength controlled molecular switch that regulated HUaa dependent intermolecular DNA bundling. Through crystallographic and solution-based studies we show that these effects mechanistically rely on HUaa promiscuity in forming multiple electrostatically driven multimerization interfaces. Changes in DNA bundling consequently affects gene expression globally, likely by constrained DNA supercoiling. Taken together our findings unveil a critical function of HU-DNA interaction in nucleoid remodeling that may serve as a general microbial mechanism for transcriptional regulation to synchronize genetic responses during the cell cycle and adapt to changing environments. (iii) We strived to elucidate how the chromosome's three-dimensional architecture is organized and maintained in bacterial cells. Using fluorescence microscopy techniques, we are probing the organization of the E. coli chromosome by directly visualizing the positions of specifically labeled DNA sites within living cells. Using two orthogonal ParB - parS systems, we were able to simultaneously label two DNA sites in two colors in the same E. coli strain. Our labeling strategy had a fixed locus as a control point in all strains, and additionally had a 'moving' locus that maps the entire chromosome in coarse-grain. Data from our experiments preliminarily suggested that there is a correlation between the linear (genetic) distance separation between DNA sites and their spatial separation. Eventually with the help of computational modeling, we hope to simulate the three-dimensional organization of the chromosome from a wealth of carefully conducted distance measurements between different DNA loci in living cells. (iv) We have additionally demonstrated separate physiological roles of specific and non-specific DNA binding of the histone-like protein HU in E. coli. A manuscript is being prepared. in bacterial physiology from maintenance of chromosome structure to regulation of gene transcription. HU is essential in many pathogens, making it an attractive target for developing anti-microbial therapeutics. A mechanistic understanding of HU DNA binding and its regulation of physiological processes will aid in the design and development of small molecule HU inhibitors. We have used Escherichia coli as a model organism to investigate how HU interacts with chromosomal DNA and regulates various physiological processes. In E. coli, HU binds to DNA in two ways: (i) with low affinity to any DNA (non-specific) through three surface-exposed lysine residues (K3, K18, and K83) that make ionic bonds with DNA phosphates; (ii) with high affinity to contorted DNA of given structures containing a pair of kinks (structure-specific) through conserved proline residues (P63) that mediate specific binding by inducing and/or stabilizing the kinks. We recently demonstrated that HU interacts with chromosomal DNA with rapid association/dissociation kinetics largely through its non-specific binding mediated by the lysine residues. This provides evidence that the overall association of HU to the chromosome is through non-specific binding. Incidentally, HU is essential in many pathogens, making it a target for developing anti-microbial drugs. A mechanistic understanding of HU DNA binding will aid in the design and development of HU inhibitors. Part B. Gene regulation in Bacteriophage Lambda and Gal operon: The current year we have made more progress in our work with phage Lambda. Investigation of RNA Polymerase & CI repressor Interactions . One of the best understood systems in genetic regulatory biology is the so called "genetic switch". This determines the choice the phage-encoded CI repressor makes by binding cooperatively to two tripartite operators, OL (OL1, OL2 & OL3) and OR (OR1, OR2 & OR3), in a defined pattern. Transcription at two lytic promoters, PL and PR, is blocked, while transcription at lysogenic promoter, PRM, is activated and repressed at low CI and high CI concentrations, respectively. The autoregulation of PRM is dependent on the interaction of RNA polymerase (RNAP) binding to the PRM promoter and CI binding to OR2. By using a purified in vitro transcription system, we analyzed the activation complex between RNAP at PRM and CI at OR2 by DNA and protein mutations. We inserted 5-bp or deleted 1-bp DNA between OR2 & OR3 to change the angular orientation and distance between RNAP and CI. We also mutated E34K of CI which interacts with RNAP during the activation of PRM. We obtained unexpected findings. First, a 1-bp DNA deletion of -34A of PRM resulted in the repression of PRM at the same CI concentration for the repression of PL and PR. This repression is depending on DNA looping and the binding of CI to OR2. Second, a 5-bp DNA insertion between the PRM promoter site and OR2 site resulted in the repression of PRM at the same CI concentration for the repression of PL and PR. Third, mutating E34K of CI which is involved in the activation complex resulted in the repression of PRM at the same CI concentration for the repression of PL and PR. Finally, DNA looping enhances PRM activation and repression. Conclusion: Disruption of the activation complex between RNAP at PRM and CI at OR2 by mutating CI or inserting or deleting base pair to change the angular orientation and distance between RNAP and CI led to the repression of PRM. These unexpected results suggest that maybe RNAP is creating negative contacts with CI at OR2 preventing RNAP from escaping and repressing PRM. Future studies are being conducted to understand the molecular mechanisms how these changes result in the repression of PRM. One attempt, currently being pursued, is to model the structure of a Prm-Ci-RNA polymerase ternary complex with wild type and different mutants DNA/proteins and inspect the feasibility of 'negative' contact between CI and RNA polymerase.