title: TrfA dimers play a role in copy-number control of RK2 replication.
abstract: Copy-number regulation of the broad-host-range plasmid RK2 is dependent on the plasmid-encoded initiator protein, TrfA, and the RK2 origin of replication. The handcuffing model for copy-number control proposes that TrfA-bound oris reversibly couple to prevent the further initiation of plasmid replication when the copy number in vivo is at or above the replicon-specific copy number. TrfA mutants have been isolated which allow for oriV replication at elevated copy numbers. To better understand the mechanism of 'handcuffing', the copy-up TrfA(G254D/S267L) mutant was characterized further. In the present study we show by size exclusion chromatography and native gel electrophoresis that unlike wt TrfA which is largely dimeric, purified His6-TrfA(G254D/S267L) is primarily monomeric. In vivo, TrfA33(G254D/S267L) supports replication of an RK2 ori plasmid in trans at a greatly elevated copy number, while in cis the plasmid exhibits runaway replication. However, expression of either of two previously isolated DNA-binding defective TrfA mutants, TrfA33(P151S) or TrfA33(S257F), in a cell transformed with a mini-RK2 replicon encoding TrfA33(G254D/S267L) results in suppression of the runaway phenotype. His6-TrfA(P151S) and His6-TrfA(S257F) purify as dimers, and when expressed in vivo are incapable of supporting RK2 plasmid replication. In contrast, combination of the trfA(P151S) or trfA(S257F) mutation with the trfA(G254D/S267L) mutations results in the expression of mutant TrfA proteins which are mainly monomers and which can no longer restore copy control to replication directed by TrfA33(G254D/S267L) in vivo. On the basis of these findings a handcuffing model is proposed, whereby oriV-bound TrfA monomers are coupled by dimeric TrfA molecules.
authors.affiliation: Center for Molecular Genetics and Department of Biology, University of California, San Diego, La Jolla, CA 92093-0322, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Toukdarian
authors.forename: D R
authors.initials: DR
authors.lastname: Helinski
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins; D018628:Gene Dosage; D016153:Genes, Suppressor; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D018741:Replication Origin
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C067405:TrfA protein, E coli