Characterizing the DNA contacts and cooperative binding of F plasmid TraM to its cognate sites at oriT.
TraM is a DNA binding protein required for conjugative transfer of the self-transmissible IncF group of plasmids, including F, R1, and R100. F TraM binds to three sites in F oriT: two high affinity binding sites, sbmA and sbmB, which are direct repeats of nearly identical sequence involved in the autoregulation of the traM gene; and a lower affinity site, sbmC, an inverted repeat important for transfer, which is situated nearest to the nic site where transfer originates. TraM bound cooperatively to its binding sites at oriT; the presence of sbmA and sbmB increased the affinity for sbmC 10-fold. Bending of oriT DNA by TraM was minimal, suggesting that TraM, a tetramer, was able to loop the DNA when bound to sbmA and sbmB simultaneously. Hydroxyl radical footprinting of DNA of sbmA and sbmC revealed that TraM contacted the DNA within a region previously delineated by DNase I footprinting. TraM protected the CT bases within the sequence CTAG, which occurred at 12-base intervals on the top and bottom strand of sbmA, most consistently with other protected bases. The footprint on sbmC revealed that the predicted inverted repeats were protected by TraM with a pattern that began at the center of the repeats and radiated outward at 11-12 base intervals toward the 5'-ends of either strand. At high protein concentrations, this pattern extended beyond the footprint defined by DNase I, suggesting that the DNA was wrapped around the protein forming a nucleosome-like structure, which could aid in preparing the DNA for transfer.
Fekete
Richard A
RA
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Frost
Laura S
LS
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D029721:Drosophila Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D017665:Hydroxyl Radical / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D015221:Potassium Channels; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D018741:Replication Origin
D001426:Bacterial Proteins; D029721:Drosophila Proteins; C102352:Ork1 protein, Drosophila; D015221:Potassium Channels; D011994:Recombinant Proteins; C074281:TraM protein, bacterial; D017665:Hydroxyl Radical; D004247:DNA; D003850:Deoxyribonuclease I