The lambda bacteriophage regulatory loop

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溶原的机制，同时保证在必要时有效地切换到裂解。这样的认识将有助于更好地控制噬菌体诱导的细菌发病机制。这也将对诱导设定值的操纵和噬菌体在基因传递应用中的使用有价值。我们将使用？噬菌体作为模型系统。最近的研究结果表明，稳定的裂解和有效的裂解转换在？依赖于抑制蛋白CI形成的DNA环。ci介导的环是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，缺乏ci介导的环的表征。这项研究的结果也将对我们理解转录调控和多蛋白介导的调控环至关重要。我们已经开始研究？我们的研究结果表明：o3位点在环形成热力学中的关键作用，环形成和破坏的复杂动力学，o3位点在帮助环形成20 nM CI方面的重要作用，o3位点在防止环破裂和最终的非特异性结合方面的重要作用，不依赖于CI浓度。综上所述，这些观察结果使我们能够提出一种新的假说，来解释这种物质形成和分解的分子机制。管理循环。这一假设表明：(i)结合在o3位点的CI二聚体在更多二聚体的“招募”中起“播种”作用，这可能促进环的形成并干扰环的破坏；（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，并确定与之相关的非环物种。我们将这样做：(i)表征不同的，相关的非环物种及其对CI浓度的依赖（原子力显微镜（AFM）和系留粒子显微镜（TPM））；（ii）量化CI非特异性结合的程度，并探索非特异性结合的CI二聚体之间协同作用的可能性（通过磁镊子和理论建模进行DNA拉力测量）。(2)阐明ci介导的环击穿机制，以及与之相关的环物种特征。我们将通过：(i)环形物种的可视化和它们的化学计量学对时间的依赖性（AFM）的表征来做到这一点；（ii）表征回路分解动力学的时间依赖性机制（AFM和TPM）。(3)研究DNA超卷曲对ci介导的环（磁镊子）的影响。该提案旨在表征λ噬菌体表观遗传开关的分子基础；对这一机制的深入了解将有助于更好地控制噬菌体诱导的细菌发病机制，并允许使用可诱导病毒进行基因传递和/或治疗。lambda开关也是远程相互作用和多蛋白组合的范例，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.