The lambda bacteriophage regulatory loop

描述（由申请人提供）：本项目的长期目标是了解确保在温和条件下维持溶原性，但在必要时保证有效转换为裂解的机制。这样的理解将是有用的，以实现更好地控制噬菌体诱导的细菌致病性。这也将是有价值的操纵的诱导的设定点和使用的基因传递应用中的诱导。我们将使用？噬菌体作为模型系统。最近的研究结果表明，无论是稳定的溶原性和有效的开关溶解？依赖于λ阻遏蛋白Cl的DNA环形成。CI介导的成环代表了最简单的转录调节反馈机制之一，并决定了噬菌体发育生长的选择。然而，缺少CI介导的成环的表征。这项研究的结果也将是我们理解转录调控和多蛋白质介导的调控环的关键。我们已经开始研究的分子机制？循环，我们的结果显示：o 3位点对于环形成的热力学的关键作用，环形成和分解的复杂动力学，o 3位点在高达20 nM CI的帮助环形成中的重要的CI浓度依赖性作用，o 3位点在防止环破裂中的重要的CI浓度独立性作用，以及最后，CI非特异性结合。总之，这些意见允许制定一个新的假说的分子机制的形成和分解的？调节环这一假设表明：（i）在α 3位点结合的Cl二聚体在“募集”更多二聚体中的“播种”作用，这可能促进环形成并干扰环分解;（ii）非特异性结合的Cl二聚体及其相互作用的生理作用。为了验证这一假设，我们提出：（1）了解CI介导的环形成机制，并确定与之相关的未成环物质，我们将通过以下方式实现这一点：（i）表征不同的、相关的未成环物质及其对CI浓度的依赖性（原子力显微镜（AFM）和系留粒子显微镜（TPM））;（ii）定量Cl非特异性结合的程度并探测非特异性结合的Cl二聚体之间的协同性的可能性（通过磁镊和理论建模的DNA牵引测量）。(2)为了阐明CI介导的环崩溃的机制，并表征与之相关的环状物质，我们将通过以下方式做到这一点：（i）环状物质的可视化和表征其化学计量对时间的依赖性（AFM）;（ii）表征负责环崩溃动力学的时间依赖性的机制（AFM和TPM）。(3)研究DNA超螺旋对CI介导的成环（磁镊）的影响。公共卫生相关性该提案旨在表征λ噬菌体中表观遗传开关的分子基础;对该机制的深刻理解将更好地控制噬菌体诱导的细菌发病机制，并允许使用诱导型病毒进行基因递送和/或治疗。λ开关也是远程相互作用和多蛋白质组装的范例，如果改变，可能导致异常和肿瘤。

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.