title: The plasmid RK2 replication initiator protein (TrfA) binds to the sliding clamp beta subunit of DNA polymerase III: implication for the toxicity of a peptide derived from the amino-terminal portion of 33-kilodalton TrfA.
abstract: The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli beta sliding clamp (beta(2)). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with beta(2) and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and beta(2) and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1. The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the beta-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent over-initiation in E. coli through its interaction with the initiator protein, DnaA, and beta(2). Our results support a model in which T1 toxicity is caused by T1 binding to beta(2), especially when T1 is overexpressed, preventing beta(2) from interacting with host replication proteins such as Hda during the early events of chromosome replication.
authors.affiliation: CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia QLD 4067, Australia. kritaya.kongsuwan@csiro.au.
authors.forename: Kritaya
authors.initials: K
authors.lastname: Kongsuwan
authors.forename: Peter
authors.initials: P
authors.lastname: Josh
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Picault
authors.forename: Gene
authors.initials: G
authors.lastname: Wijffels
authors.forename: Brian
authors.initials: B
authors.lastname: Dalrymple
mesh_terms: D000595:Amino Acid Sequence; D002455:Cell Division; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology*; D021122:Protein Subunits / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D021122:Protein Subunits; C067405:TrfA protein, E coli; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III