Molecular Conversion and Function Exmression of Replication Initiator Protein

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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