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Pseudomonas: Biology and Diversity |
Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain1
Author for correspondence: Víctor de Lorenzo. Tel: +34 91 585 4536. Fax: +34 91 585 4506. e-mail: vdlorenzo{at}cnb.uam.es
The ATP-dependent multimerization process undergone by the 
54-dependent activator XylR of the TOL plasmid pWW0 of Pseudomonas putida when bound to the upstream activating sequences (UAS) of the cognate Pu promoter was examined by transmission electron microscopy (TEM). To this end, supercoiled DNA templates were combined with increasing concentrations of the constitutive XylR variant XylR
A, with or without ATP or its non-hydrolysable analogue ATP
S, and the resulting complexes were visualized by TEM. The different types of DNA–protein association were analysed and a statistical study of the frequency of the various forms was made. ATP appeared to establish an equilibrium between different molecular associations, as well as major changes in the physical shape of the DNA–protein complexes. The formation of higher nucleoprotein structures frequently bearing DNA bends became manifest. Such complexes often engaged otherwise separated UAS-containing plasmids, indicating that the ATP-driven multimer included XylR molecules recruited in trans. Whilst ATP caused the different types of XylR–DNA complex to occur at quite balanced frequencies, ATPS 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 transcriptional activation complex is disassembled.
Keywords: Pseudomonas putida, TOL plasmid, Pu promoter, sigma 54, enhancers
Abbreviations: TEM, transmission electron microscopy; UAS, upstream activating sequences
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