Topology of a Membrane Associated Regulator of Prokaryotic DNA replication

摘要

This proposal has focused on a broad host range plasmid, RK2, as a model system to study how a pair of initiation proteins encoded by the plasmid for DNA replication function when replication occurs associated with the membrane of its bacterial host Escherichis coli. It was found that the initiation proteins were very resistant to agents that dissociate weakly bound (peripheral) membrane associated proteins. In this respect, they resemble strongly bound (integral) membrane proteins. Yet, there is only one short region of hydrophobic amino acids that might be involved in such membrane association. To determine the topology of these proteins in the membrane, additional studies involving site directed mutagenesis of the small domain, and the construction of protein fragments demonstrated not only that the small region was essential for membrane binding but also for viability of the plasmid. In addition, two other membrane binding domains were detected, one near the beginning (amino terminal) of the protein, the other, adjacent to the short hydrophobic domain. Finally, anionic phospholipids (part of the membrane) may be involved in maintaining plasmid viability by anchoring the initiation proteins to the membrane.