Ultracentrifugation

Different oligomeric states of the p73 DBD was identified by analytical ultracentrifugation. The isolated DBD was shown to exist as a soluble monomer in the absence of DNA, which could happen after translation or prior to nuclear import.

Figure 3. p73 DBD tetramerisation stabilized by surface loops.
Zinc ion coordination is enlarged on mouseover.

Remarkably, the presence of ~12bp half-site RE in the centrifugation mixture resulted in the sedimentation coefficient roughly twice as large, indicating that DBD dimerisation had occurred.

Furthermore, when ~20bp sequence containing the full RE was added, the coefficient was increased to almost double again – implying DBD tetramerisation had occured.

Figure 3 shows the crystal structure of p73 DBD tetramer bound to a DNA fragment carrying full RE. Its association is suggested to be stabilized by surface loops with great conformational flexibility, as well as hydrogen bonding and hydrophobic interactions.

 

The animation below shows the sequential process of p73 tetramerization.