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)-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 XylRDeltaA, with or without ATP or its non-hydrolysable analogue ATPgammaS, 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, ATPgammaS 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.
Garmendia
Junkal
J
Centro Nacional de Biotecnologı́a CSIC, Campus de Cantoblanco, 28049 Madrid, Spain1.
de Lorenzo
Vı Ctor
VC
Centro Nacional de Biotecnologı́a CSIC, Campus de Cantoblanco, 28049 Madrid, Spain1.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D008854:Microscopy, Electron / Q000379:methods; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C074322:XylR protein, Pseudomonas; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54