Visualization of DNA-protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida

The ATP-dependent multimerization process undergone by the sigma (54)-depen dent activator XyIR of the TOL plasmid pWW0 of Pseudomonas putida when boun d to the upstream activating sequences (UAS) of the cognate Pu promoter was examined by transmission electron microscopy (TEM). To this end, supercoil ed DNA templates were combined with increasing concentrations of the consti tutive XyIR variant XyIR DeltaA, with or without AIP or its non-hydrolysabl e analogue ATP gammaS, and the resulting complexes were visualized by TEM. The different types of DNA-protein association were analysed and a statisti cal study of the frequency of the various forms was made. ATP appeared to e stablish an equilibrium between different molecular associations, as well a s major changes in the physical shape of the DNA-protein complexes. The for mation of higher nucleoprotein structures frequently bearing DNA bends beca me manifest. Such complexes often engaged otherwise separated UAS-containin g plasmids, indicating that the ATP-driven multimer included XyIR molecules recruited in trans. Whilst ATP caused the different types of XyIR-DNA comp lex to occur at quite balanced frequencies, ATP gammaS appeared to displace the distribution predominantly towards the higher order forms. These data are compatible with the notion that each time ATP is hydrolysed the transcr iptional activation complex is disassembled.