Regulation of Gene Transcription

染色体的结构和功能。从我们对基因调控机制的研究中，我们先前提出细菌染色体（类核）具有条件依赖的定义结构，该结构决定基因表达。HU是真细菌中最保守的核相关蛋白，但它如何影响整体染色体组织和基因表达尚不清楚。(i)使用单分子跟踪，我们证明了HU与染色体DNA表现出非特异性、弱和短暂的相互作用。这些相互作用主要是由三个保守的，表面暴露的赖氨酸残基（triK）介导的，这些残基先前被证明是与DNA非特异性结合的原因。在HUa（triKA）突变体中，这些弱的、短暂的相互作用的丧失导致了过凝聚和错误分离的类核。突变HUa亚基中的保守脯氨酸残基（P63A），删除HUb亚基，或删除核相关的naRNAs，这些都与HU与扭曲或十字形DNA的高亲和力结合有关，与HUa（triKA）突变体相比，导致HU的相互作用动力学变化较小，但高度扩展的类核。我们的研究结果表明，HU通过与染色体DNA的差异相互作用，在维持适当的类核体积方面发挥双重作用。一方面，HU通过特定的DNA结构结合相互作用使类核紧密。另一方面，它通过许多非特异性的、弱的和短暂的与体染色体的相互作用使类核去致密化。这种动态相互作用可能有助于细菌类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射线断层扫描对接近天然的、未标记的大肠杆菌细胞进行成像，我们发现HU通过促进由较少凝聚的孤立结构域包围的致密凝聚核的形成来重塑类核。细胞生长过程中的类核重塑和环境适应与pH和离子强度控制的分子开关相关，这些分子开关调节了依赖于HUaa的分子间DNA捆绑。通过晶体学和基于溶液的研究，我们发现这些效应在形成多个静电驱动的多聚界面时，机制上依赖于华化华杂性。因此，DNA捆绑的变化影响了基因的整体表达，可能是受约束的DNA超卷曲。综上所述，我们的发现揭示了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，在细胞周期中同步遗传反应并适应不断变化的环境。（iii）我们努力阐明染色体的三维结构是如何在细菌细胞中组织和维持的。利用荧光显微镜技术，我们通过直接观察活细胞内特定标记的DNA位点的位置来探测大肠杆菌染色体的组织。利用两个正交的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。我们的标记策略在所有菌株中都有一个固定的位点作为控制点，另外还有一个“移动”的位点，以粗粒方式绘制整个染色体。我们的实验数据初步表明，DNA位点之间的线性（遗传）距离分离与其空间分离之间存在相关性。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位点之间的距离进行大量仔细测量，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在许多病原体中是必需的，使其成为开发抗微生物疗法的一个有吸引力的靶点。对HU DNA结合及其对生理过程的调控机制的理解将有助于设计和开发小分子HU抑制剂。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以两种方式与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，与任何DNA（非特异性）具有低亲和力；（ii）对含有一对扭结（结构特异性）的给定结构的扭曲DNA具有高亲和力，通过保守的脯氨酸残基（P63）通过诱导和/或稳定扭结介导特异性结合。我们最近证明，HU与染色体DNA的相互作用具有快速的结合/解离动力学，主要是通过赖氨酸残基介导的非特异性结合。这证明了HU与染色体的整体关联是通过非特异性结合。顺便说一句，HU在许多病原体中是必需的，使其成为开发抗微生物药物的靶点。对胡DNA结合机制的理解将有助于胡抑制剂的设计和开发。第二部分：噬菌体Lambda和Gal操纵子的基因调控：这一年我们在噬菌体Lambda的研究上有了新的进展。RNA聚合酶与CI抑制因子相互作用的研究。在遗传调控生物学中，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抑制因子通过以确定的模式与两个三方操作符OL （OL1， OL2和OL3）和OR （OR1， OR2和OR3）协同结合而做出的选择。两个裂解启动子PL和PR的转录被阻断，而在低CI和高CI浓度下，溶原启动子PRM的转录分别被激活和抑制。PRM的自动调节依赖于RNA聚合酶（RNAP）与PRM启动子结合和CI与OR2结合的相互作用。通过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RNAP和OR2处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的DNA，以改变RNAP与CI之间的角取向和距离。我们还突变了在PRM激活过程中与RNAP相互作用的CI的E34K。我们得到了意想不到的发现。首先，在相同CI浓度下，PRM -34A的1 bp DNA缺失导致PRM对PL和PR的抑制。这种抑制依赖于DNA环和CI与OR2的结合。其次，在PRM启动子位点和OR2位点之间插入5bp的DNA，导致相同CI浓度的PRM受到抑制，从而抑制PL和PR。第三，参与激活复合物的CI的E34K突变，导致相同CI浓度的PRM受到抑制，从而抑制PL和PR。最后，DNA环化增强了PRM的激活和抑制。结论：通过突变CI或插入或删除碱基对改变RNAP与CI之间的角度取向和距离，破坏RNAP在PRM和OR2位点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可能在OR2位点与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到*截断*

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 *TRUNCATED*