Molecular Conversion and Function Exmression of Replication Initiator Protein

RepE是大肠杆菌中F质粒的DNA复制起始蛋白，有两种分子结合状态，即单体和二聚体。根据其分子结合状态的不同，RepE的分子功能也有本质的不同。该单体起DNA复制启动子的作用，与F质粒的复制起点结合。另一方面，优势二聚体作为转录抑制因子，通过对启动子蛋白的负反馈调节，结合到epE基因的启动子/操纵子区域，抑制F质粒的复制。本研究的目的是通过对RepE的惰性二聚体形式和活性单体形式的结构比较，揭示RepE蛋白是如何作为DNA复制启动子被激活的。由于没有RepE二聚体的结构信息，我们已经开始了对这种二聚体状态的X射线结晶学研究。我们成功地制备了t-…的结晶样品更多的RepE二聚体与RepE操纵子DNA经过多次试验，得到了适合于X射线工作的RepE-DNA络合物的晶体。用同步辐射收集衍射数据，以结构已确定的RepE突变体RepE54的单体结构为搜索模型进行分子置换。二聚体与单体的结构比较显示出一些特征。尽管单体和二聚体之间N-末端和C-末端结构域的构象基本相同，但连接这两个结构域的结构域连接体的二级结构和相对结构域取向发生了显著变化。此外，在RepE的结构域连接区内，预测了F质粒复制所必需的DNAK和DNAJ蛋白的相互作用部位，属于DNAK伴侣系统。这些发现表明，当DNAK伴侣与RepE结合时，RepE结构域连接区的局部结构变化将导致RepE二聚体的结构域重排和解离，从而激活RepE作为复制启动子。为了详细了解RepE和伴侣之间的相互作用并支持所提出的激活机制，我们还试图制备RepE-伴侣复合体的样品和一些RepE或伴侣的突变体。较少

RepE, the DNA replication initiator protein of the F plasmid in Escherichia coli, has two molecular association states, i.e., monomer and dimer. Depending on its molecular association state, the molecular function of RepE is intrinsically different. The monomer functions as a DNA replication initiator, which binds to a replication origin of the F plasmid. On the other hand, the dominant dimer plays as a transcriptional repressor, which binds to the promoter/operator region of the repE gene to inhibit the F plasmid replication by negative feedback regulation of the initiator protein. The aim of this study is to reveal how the RepE protein is activated as a DNA replication initiator by means of a structural comparison between the inert dimeric form and the active monomeric form of RepE. Because no structural information of the RepE dimer was available, we have started X-ray crystallographic study of this dimeric state. We have succeeded in the preparation of a crystallization sample of t … More he RepE dimer in complex with the repE operator DNA as a result of many trials, and obtained crystals of the RepE-DNA complex suitable for X-ray work. Diffraction data were collected with synchrotron radiation, and the molecular replacement was employed using the monomeric structure of a RepE mutant, RepE54, whose structure had already been determined, as a search model. Structural comparison of the dimer with the monomer showed some characteristic features. Although the conformations of two functional domains, N- and C-terminal domains, are essentially the same between the monomer and dimer, the secondary structure of a domain linker connecting the two domains and the relative domain orientation are significantly changed. Moreover, interaction sites of DnaK and DnaJ proteins belonging to the DnaK chaperone system, which is necessary for the F plasmid replication, are predicted within the domain linker region of RepE. These findings suggest that a local structural alteration in the domain linker region of RepE upon binding of the DnaK chaperones will cause domain rearrangement and dissociation of the RepE dimer, resulting in activation of RepE as a replication initiator. To recognize interactions between RepE and chaperones in detail and to support the proposed activation mechanism, we have also tried to prepare a sample of the RepE-chaperone complex and some mutants of RepE or chaperones. Less