The essential transfer protein TraM binds to DNA as a tetramer

The TraM proteins encoded by F-like plasmids are sequence specific DNA bind ing proteins that are essential for conjugative DNA transfer. We investigat ed the quarternary structure and the DNA binding properties of the TraM wil d-type protein of the resistance plasmid R1 and two mutant forms thereof. S ize-exclusion chromatography and differential scanning calorimetry showed t hat purified TraM protein (amino acids 2-127) forms stable tetramers in sol ution. A truncated version of the protein termed TraMM26 (amino acids 2-56) forms dimers, Thus, the dimerization and tetramerization domains can be as signed to the N-terminal and C-terminal domains of TraM, respectively. Furt her 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 pr otein is also tetrameric when bound to its minimal DNA binding site, The de duced binding constant in the range of 10(8) M-1 demonstrated a very strong binding of TraM to its preferred DNA sequence. Secondary structure analysi s based on CD measurements showed that TraM is mainly cu-helical with a sig nificant increase in alpha-helicity (48 to 58%) upon DNA-binding, indicatin g an induced fit mechanism.