In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.
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.
Pansegrau
W
W
Max-Planck-Institut für Molekulare Genetik, Abteilung Schuster, Berlin, Federal Republic of Germany.
Balzer
D
D
Kruft
V
V
Lurz
R
R
Lanka
E
E
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
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