Interactions among GalR-binding sites on bacterial DNA

我们对细菌中DNA包装的理解仍然不清楚，因为还没有描述像真核生物中那样的统一机制。此外，许多转录事件需要DNA上的远程位点以明确的配置聚集在一起。这些事件背后的机制和可能的辅助蛋白尚不清楚。已知GalR蛋白与DNA操纵子结合，在大肠杆菌中完成半乳糖基因调控，在另一种蛋白质HU的存在下甚至更强，HU是已知的影响DNA弯曲的蛋白质。大肠杆菌中这两种蛋白质的丰富及其染色体上多个GalR结合位点的存在，支持了GalR是连接远程DNA位点的中心角色的假设，从而发挥了DNA包装剂和转录事件辅助蛋白质的双重作用。 采用了生化和成像相结合的方法。荧光标记的GalR在体内被可视化，并且GalR与不同大小的超螺旋质粒并携带多个GalR结合位点的络合物在单分子水平上被可视化。对于后者，我们使用原子力显微镜，我们可以清楚地看到蛋白质结合的效果。对蛋白质结合前后环大小的测量清楚地支持了这样的假设，即不同大小的GalR寡聚体连接了远程DNA位点。

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.