In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.
Proceedings of the National Academy of SciencesPublished 1 September 1990Open access
Werner Pansegrau, D Balzer, Volker Kruft, Rudi Lurz, Erich Lanka
Citations146
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TL;DR
A complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmids for generating the single strand to be transferred.
Abstract
During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT. The structure can be visualized on electron micrographs. Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. 265, 10637-10644]. Nicking and relaxosome formation require supercoiled DNA. Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.
Keywords
Biochemistry, Genetics and Molecular Biology
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