Many key metabolic reactions are controlled or influenced by components that are associated with the cell membrane of prokaryotes. A vital reaction, which has emerged as subject to such control, is DNA replication. Of many reasons for this control, the lack of a nucleus in prokaryotes has required the genetic material to seek a cellular site where important events involved in its replication and segregation into a daughter cell could be sequestered. A great deal of evidence has been presented to support this supposition and the replicon model of Jacob et AL (1963, Cold Spring Harbor Symp. Quant. Biol. 28:329-348) still represents a good conceptual framework (with modifications) to explain how such replication and segregation are linked by the cell membrane. The proposed investigations will continue to focus on a remarkable pair of initiation proteins (TrfA) encoded by overlapping genes from a medically important broad host range plasmid RK2 cultured in its E. coli host. These proteins (33 and 43 kDa) express what appears to be a fundamental paradox, that of interacting in a """"""""soluble"""""""" environment (namely the cytoplasm) with the origin region of plasmid replication (ORIV) while being sequestered in a hydrophobic environment (namely the membrane) so as to anchor them to a stable site. A critical region near the carboxyl terminus of both proteins consisting of hydrophobic amino acids (HR) was identified as an important domain for membrane interaction and plasmid viability. Other regions, however, are also involved in membrane binding, although there is no obvious amphipathic helix or hydropathicity for them. One such region is not only associated with the amino terminal portion of the 33 kDA species but also has a synthetic lethality for the host cell indicating an interaction with a host component. These results will be extended in two major directions. 1) an attempt to detect an interaction of the TrfA proteins with a host component by multicopy suppressor analysis and 2) demonstrate that a specific submembrane domain of the E. coli host represents the actual site for plasmid DNA replication and the embodiment of the replicon model for prokaryotic DNA replication.
| Kim, Peter D; Banack, Trevor; Lerman, Daniel M et al. (2003) Identification of a novel membrane-associated gene product that suppresses toxicity of a TrfA peptide from plasmid RK2 and its relationship to the DnaA host initiation protein. J Bacteriol 185:1817-24 |
