The Essential Transfer Protein TraM Binds to DNA as a Tetramer

Abstract The TraM proteins encoded by F-like plasmids are sequence specific DNA binding proteins that are essential for conjugative DNA transfer. We investigated the quarternary structure and the DNA binding properties of the TraM wild-type protein of the resistance plasmid R1 and two mutant forms thereof. Size-exclusion chromatography and differential scanning calorimetry showed that purified TraM protein (amino acids 2–127) forms stable tetramers in solution. A truncated version of the protein termed TraMM26 (amino acids 2–56) forms dimers. Thus, the dimerization and tetramerization domains can be assigned to the N-terminal and C-terminal domains of TraM, respectively. Further analyses using chemical cross-linking and light scattering corroborated the preferentially tetrameric nature of the protein but also suggest that TraM has a tendency to form higher aggregates. Band-shift and fluorescence spectroscopy investigations of TraM-DNA complexes revealed that the TraM protein is also tetrameric when bound to its minimal DNA binding site. The deduced binding constant in the range of 108 m −1 demonstrated a very strong binding of TraM to its preferred DNA sequence. Secondary structure analysis based on CD measurements showed that TraM is mainly α-helical with a significant increase in α-helicity (48 to 58%) upon DNA-binding, indicating an induced fit mechanism.