title: In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.
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.
authors.affiliation: Max-Planck-Institut für Molekulare Genetik, Abteilung Schuster, Berlin, Federal Republic of Germany.
authors.forename: W
authors.initials: W
authors.lastname: Pansegrau
authors.forename: D
authors.initials: D
authors.lastname: Balzer
authors.forename: V
authors.initials: V
authors.lastname: Kruft
authors.forename: R
authors.initials: R
authors.lastname: Lurz
authors.forename: E
authors.initials: E
authors.lastname: Lanka
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009698:Nucleoproteins / Q000378:metabolism* / Q000648:ultrastructure; D011815:R Factors; D015183:Restriction Mapping
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D009687:Nuclear Proteins; D009698:Nucleoproteins; C065598:TraH protein, Bacteria; C488999:TraJ protein, E coli; C041427:TraJ protein, bacteria; C027093:TraI protein, E coli; D004265:DNA Helicases