Interactions among GalR-binding sites on bacterial DNA

细菌 DNA 上 GalR 结合位点之间的相互作用

• 批准号: 8340643
• 负责人: EMILIOS K DIMITRIADIS
• 金额: $ 1.28万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8340643
• 关键词: Atomic Force Microscopy; Bacteria; Bacterial DNA; Binding Sites; Biochemical; Chromosomes; Complex; DNA; DNA Binding; DNA Packaging; Escherichia coli; Eukaryota; Event; Gal repressor protein; Galactose; Gene Expression Regulation; Genes; Genetic Transcription; Goals; HU Protein; Measurement; Plasmids; Process; Protein Binding; Proteins; Role; Site; Transcription Process; imaging modality; in vivo; single molecule

Our understanding of DNA packaging in bacteria remains obscure, as no unifying mechanism, such as that in eukaryotes, has been described. In addition, many transcription events require that remote sites on the DNA come together in a well defined configuration. The mechanism behind such events and the possible ancillary proteins are not known. The GalR protein is known to bind DNA operator sites to accomplish galactose gene regulation in E-coli, and even more strongly in the presence of another protein, HU, which is known to effect DNA bending. The abundance of both proteins in E-coli and the existence of multiple GalR binding sites on its chromosome, motivate the hypothesis that GalR is a central actor in bridging remote DNA sites, thus acting in a dual role of a DNA packaging agent and of a protein ancillary to transcription events. A combination of biochemical and imaging methods are employed. Fluorescently tagged GalR are visualized in vivo and complexes of GalR with supercoiled plasmids of various sizes and carrying a number of GalR binding sites are visualized at a single molecule level. We use atomic force microscopy for the latter and we can clearly see the effects of protein binding. Measurements of loop sizes before and after protein binding clearly support the hypothesis that GalR oligomers of various sizes bridge remote DNA sites.

我们对细菌中DNA包装的理解仍然模糊，因为没有统一的机制，如真核生物中的机制，已经被描述。 此外，许多转录事件需要DNA上的远程位点以明确定义的构型聚集在一起。 这些事件背后的机制和可能的辅助蛋白尚不清楚。 已知GalR蛋白结合DNA操纵子位点以实现大肠杆菌中的半乳糖基因调控，并且在另一种已知影响DNA弯曲的蛋白HU存在下甚至更强烈。 这两种蛋白质在大肠杆菌中的丰度和在其染色体上存在多个GalR结合位点，激发了GalR是桥接远程DNA位点的中心演员的假设，从而起DNA包装剂和辅助转录事件的蛋白质的双重作用。 采用生物化学和成像方法的组合。标记的GalR在体内可视化，GalR与各种大小的超螺旋质粒的复合物，并携带许多GalR结合位点，在单分子水平可视化。我们使用原子力显微镜观察后者，我们可以清楚地看到蛋白质结合的效果。蛋白质结合前后的环大小的测量清楚地支持以下假设，即各种大小的GalR寡聚体桥接远程DNA位点。