The lambda bacteriophage regulatory loop

lambda噬菌体调节环路

• 批准号: 8072532
• 负责人: Laura Finzi
• 金额: $ 25.83万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072532
• 关键词: Acute; Address; Affinity; Atomic Force Microscopy; Bacteriophage lambda; Bacteriophages; Binding; Biological Models; Cell physiology; Chromatin Loop; Chromosomal translocation; Chromosomes, Human, Pair 3; Complex; Cytolysis; DNA; DNA-Directed RNA Polymerase; Dependence; Dependency; Development; Drug Formulations; Ensure; Epigenetic Process; Feedback; Gene Delivery; Genes; Genetic Recombination; Genetic Transcription; Goals; Growth; Health; Homeostasis; Imagery; Kinetics; Lead; Left; Lysogeny; Lytic; Magnetism; Maintenance; Measurement; Mediating; Microscopy; Molecular; Operator Regions; Outcomes Research; Pathogenesis; Physiological; Proteins; Repression; Repressor Proteins; Research Design; Research Methodology; Role; Rupture; Site; Superhelical DNA; Telomere Maintenance; Testing; Theoretical model; Thermodynamics; Time; Transcriptional Regulation; Viral; Virus; Work; base; dimer; lambda repressor; novel; particle; prevent; promoter; research study; stoichiometry; tumor

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the mechanism that ensures lysogeny maintenance in temperate phages yet guaranteeing efficient switch to lysis when necessary. Such understanding will be useful in order to achieve a better control of phage-induced bacterial pathogenesis. It will also be valuable for manipulation of the inducibility set-point and use of phages in gene delivery applications. We will use ? bacteriophage as a model system. Recent findings showed that both stable lysogeny and efficient switch to lysis in ? rely on DNA loop formation by the lambda repressor protein CI. CI-mediated looping represents one of the simplest transcriptional regulatory feedback mechanisms and determines the choice of developmental growth by the phage. However, a characterization of CI-mediated looping is missing. The outcome of this research will also be pivotal for both our understanding of transcriptional regulation and multi-protein-mediated regulatory loops. We have started investigating the molecular mechanism of ? looping and our results show: a pivotal role of the o3 sites for the thermodynamics of loop formation, a complex kinetics for both loop formation and breakdown, an important, CI concentration-dependent role of the o3 sites in aiding loop formation up to 20 nM CI, an important, CI concentration-independent role of the o3 sites in preventing loop rupture and, finally, CI non-specific binding. Together, these observations allow the formulation of a new hypothesis about the molecular mechanism for the formation and breakdown of the ? regulatory loop. This hypothesis suggests: (i) a "seeding" role for the CI dimers bound at the o3 sites in the "recruitment" of more dimers which may facilitate loop formation and interfere with loop breakdown; (ii) a physiological role for non-specifically bound CI dimers and their interaction. To test this hypothesis, we propose: (1) To understand the mechanism of CI-mediated loop formation and to identify the unlooped species relevant to it. We will do this by: (i) characterizing the different, relevant unlooped species and their dependence on CI concentration (Atomic Force Microscopy (AFM) and Tethered Particle Microscopy(TPM)); (ii) quantifying the extent of CI nonspecific binding and probing the possibility of cooperativity between non-specifically bound CI dimers (DNA pulling measurements by magnetic tweezers and theoretical modeling). (2) To elucidate the mechanism of CI-mediated loop breakdown, and characterization of the looped species relevant to it. We will do this by: (i) visualization of looped species and characterization of the dependence of their stoichiometry on time (AFM); (ii) characterization of the mechanism responsible for the time dependency of the kinetics of loop breakdown (AFM and TPM). (3) To investigate the effect of DNA supercoiling on CI-mediated looping (magnetic tweezers). PUBLIC HEALTH RELEVANCE The proposal aims to characterize the molecular bases of the epigenetic switch in lambda bacteriophage; an acute understanding of this mechanism will provide a better control of phage-induced bacterial pathogenesis and allow the use of inducible viruses for gene delivery and/or therapy. The lambda switch is also a paradigm of long-range interactions and multi-protein assemblages which if altered can lead to anomalies and tumors.

描述（由申请人提供）：该项目的长期目标是了解确保在温带噬菌体中维持裂解的机制，并在必要时确保有效切换到裂解。这样的理解将有用，可以更好地控制噬菌体诱导的细菌发病机理。这也将对于操纵诱导性设定点和在基因递送应用中的使用有价值。我们会使用吗？噬菌体作为模型系统。最近的发现表明，稳定的溶疗和有效切换到裂解中？依靠Lambda抑制剂蛋白CI的DNA环形成。 CI介导的循环代表了最简单的转录调节反馈机制之一，并决定了通过噬菌体选择发育生长的选择。但是，缺少CI介导的循环的表征。这项研究的结果对于我们对转录调控和多蛋白介导的调节环的理解也将是关键的。我们已经开始研究分子机制？循环和我们的结果表明：循环形成热力学的O3位点的关键作用，这是环路形成和分解的复杂动力学，这是O3位点在辅助环路中的重要，CI浓度依赖性的作用CI是O3位点在防止环形破裂和CI非特异性结合中的重要的CI浓度独立的作用。总之，这些观察结果允许制定有关形成和分解的分子机制的新假设？监管循环。该假设表明：（i）在更多二聚体的“募集”中绑定的CI二聚体的“播种”作用，这可能有助于循环形成并干扰循环分解； （ii）非特异性结合的CI二聚体及其相互作用的生理作用。为了检验这一假设，我们建议：（1）了解CI介导的环形成的机理，并确定与之相关的无环物种。我们将通过：（i）表征不同的，无环的物种及其对CI浓度（原子力显微镜（AFM）和束缚粒子显微镜（TPM））的依赖性； （ii）量化CI非特异性结合的程度，并探测非特异性结合的CI二聚体（通过磁性镊子和理论建模的DNA拉值测量）之间合作的可能性。 （2）阐明了CI介导的环形分解的机理，并表征了与之相关的环形物种。我们将通过：（i）可视化循环物种的可视化以及其化学计量法对时间（AFM）的依赖性的表征； （ii）表征导致环路分解动力学（AFM和TPM）时间依赖性的机制。 （3）研究DNA超螺旋对CI介导的环（磁镊）的影响。公共卫生相关性该提案旨在表征lambda噬菌体表观遗传转换的分子碱基。对这种机制的急性理解将更好地控制噬菌体诱导的细菌发病机理，并允许将诱导病毒用于基因递送和/或治疗。 Lambda开关也是远距离相互作用和多蛋白组合的范式，如果改变会导致异常和肿瘤。