The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone.
Initiation and control of replication of the broad-host-range plasmid RK2 requires two plasmid-encoded elements, the replication origin (oriV) and the initiation protein TrfA. Purified TrfA is largely in the form of a dimer; however, only the monomeric form of the protein can bind specifically to the direct repeats (iterons) at the RK2 origin. The largely dimeric form of wild-type TrfA is inactive in the initiation of replication of RK2 in an in vitro replication system reconstituted from purified components. However, preincubation of the TrfA protein with the ClpX molecular chaperone isolated from Escherichia coli activates the initiator protein for replication in the purified system. We further observed that ClpX, in an ATP-dependent reaction, greatly increases the proportion of TrfA monomers and, therefore, the ability of this protein to bind to iterons localized within RK2 origin. Finally, a copy-up mutant of the TrfA protein which is largely in the monomer form is active in the reconstituted in vitro replication system, and its activity is not affected by ClpX.
Konieczny
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Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634, USA.
Helinski
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D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D018832:Molecular Chaperones / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D012697:Serine Endopeptidases / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C067405:TrfA protein, E coli; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities