This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kongsuwan, K. Articles by Dalrymple, B. Search for Related Content PubMed PubMed Citation Articles by Kongsuwan, K. Articles by Dalrymple, B.

 Previous Article  |  Next Article 

Journal of Bacteriology, August 2006, p. 5501-5509, Vol. 188, No. 15
0021-9193/06/$08.00+0     doi:10.1128/JB.00231-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Plasmid RK2 Replication Initiator Protein (TrfA) Binds to the Sliding Clamp ß Subunit of DNA Polymerase III: Implication for the Toxicity of a Peptide Derived from the Amino-Terminal Portion of 33-Kilodalton TrfA

CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia QLD 4067, Australia,¹ Ecole de Biologie Industrielle, 32, Boulevard du Port, 95094 Cergy-Pontoise Cedex, France²

Received 14 February 2006/ Accepted 3 May 2006

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 ß sliding clamp (ß₂). 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 ß₂ and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and ß₂ and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1 (P. D. Kim, T. M. Rosche, and W. Firshein, Plasmid 43:214-222, 2000). The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the ß-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent overinitiation in E. coli through its interaction with the initiator protein, DnaA, and ß₂. Our results support a model in which T1 toxicity is caused by T1 binding to ß₂, especially when T1 is overexpressed, preventing ß₂ from interacting with host replication proteins such as Hda during the early events of chromosome replication.