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-...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1990-09, Vol.87 (17), p.6555-6559
Main Authors: Pansegrau, Werner, Balzer, Dietmar, Kruft, Volker, Lurz, Rudi, Lanka, Erich
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Outer Membrane Proteins - genetics
Bacterial Outer Membrane Proteins - metabolism
Bacterial Proteins
Base Sequence
Cloning, Molecular
Conjugation, Genetic
DNA
DNA Helicases - genetics
DNA Helicases - metabolism
DNA, Superhelical - genetics
DNA, Superhelical - ultrastructure
Escherichia coli - genetics
Escherichia coli Proteins
Gels
Genes
Molecular Sequence Data
Nuclear Proteins - genetics
Nuclear Proteins - metabolism
Nucleoproteins
Nucleoproteins - metabolism
Nucleoproteins - ultrastructure
Nucleotide sequences
Operator regions
Operons
Plasmids
Proteins
R Factors
Restriction Mapping
Start codon
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.87.17.6555