| Many host- and plasmid-encoded proteins are required to efficiently transfer the Escherichia coli F plasmid. The latter are divided into five functional groups: pilus synthesis and assembly, surface exclusion, mating aggregate stabilization, gene regulation, and DNA metabolism. Proteins of the last group are TraI, TraY, TraD, and TraM. TraI is a relaxase and helicase, and requires IHF and TraY, which bind to oriT, for cleavage. TraD is an inner membrane protein and is thought to link oriT-bound proteins to the transfer apparatus in the membrane. TraM binds to three sites in oriT, and is required for transfer, but not for cleavage in vitro. Two of the TraM binding sites, sbmA and sbmB, autoregulate traM transcription, and the last site, sbmC, is more important for transfer.; Nicking assays performed on an F traM mutant show that TraM is not required for relaxase activity in vivo. Nicking and mobilization assays using chimeric plasmids constructed from F and the F-like plasmid R100-1, using F or R100-1 Tra proteins provided in trans, suggest that protein:DNA interactions at oriT provide much of the previously characterized plasmid specificity. Biochemical characterization of TraM indicate that TraM exists mainly as tetramers in solution, but dimers and tetramers bind to TraM binding sites. Experiments involving the yeast two-hybrid system identified two domains which participate in TraM multimerization, a central and a carboxyl-terminal domain. Deleting various regions of TraM in the TraM:GAL4 fusions suggests that each domain interacts with a similar domain in another molecule to form TraM multimers.; EMSA experiments determined that TraM binds to its binding sites cooperatively and these complexes are very stable once bound. Hydroxyl radical footprinting defined which bases are protected by the bound protein.; Using this data, the factors responsible for relaxation and transfer mutant phenotypes are summarized. The identity of the domain responsible for tetramerization of TraM is also proposed based on similarities to the well-characterized LacR protein. Finally, a model describing the mechanism by which TraM binds to oriT is formulated based on data from EMSA, sizing of DNA bound proteins, and hydroxyl radical footprinting. |
