1887

Abstract

The ATP-dependent multimerization process undergone by the σ-dependent activator XylR of the TOL plasmid pWW0 of when bound to the upstream activating sequences (UAS) of the cognate promoter was examined by transmission electron microscopy (TEM). To this end, supercoiled DNA templates were combined with increasing concentrations of the constitutive XylR variant XylRΔA, with or without ATP or its non-hydrolysable analogue ATPγS, 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 . Whilst ATP caused the different types of XylR–DNA complex to occur at quite balanced frequencies, ATPγS 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.

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2000-10-01
2019-10-21
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References

  1. Abril, M. A., Buck, M. & Ramos, J. L. ( 1991; ). Activation of the Pseudomonas TOL plasmid upper pathway operon. J Biol Chem 266, 15832-15838.
    [Google Scholar]
  2. Assinder, S. J. & Williams, P. A. ( 1990; ). The TOL plasmids: determinants of the catabolism of toluene and xylenes. Adv Microb Physiol 31, 1-69.
    [Google Scholar]
  3. Delgado, A. & Ramos, J. L. ( 1994; ). Genetic evidence for activation of the positive transcriptional regulator XylR, a member of the NtrC family of regulators, by effector binding. J Biol Chem 269, 8059-8062.
    [Google Scholar]
  4. Drummond, M., Whitty, P. & Wooten, J. ( 1986; ). Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J 5, 441-447.
    [Google Scholar]
  5. Férnandez, S., de Lorenzo, V. & Pérez-Martı́n, J. ( 1995; ). Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains. Mol Microbiol 16, 205-213.[CrossRef]
    [Google Scholar]
  6. Inouye, S., Nakazawa, A. & Nakazawa, T. ( 1988; ). Nucleotide sequence of the regulatory gene xylR of the TOL plasmid from Pseudomonas putida. Gene 66, 301-306.[CrossRef]
    [Google Scholar]
  7. Li, J., Passaglia, L., Rombel, I., Yan, D. & Kustu, S. ( 1999; ). Mutations affecting motifs of unknown function in the central domain of nitrogen regulatory protein C. J Bacteriol 181, 5443-5454.
    [Google Scholar]
  8. de Lorenzo, V., Herrero, M., Metzke, M. & Timmis, K. N. ( 1991; ). An upstream XylR- and IHF-induced nucleoprotein complex regulates the σ54-dependent Pu promoter of TOL plasmid. EMBO J 10, 1159-1167.
    [Google Scholar]
  9. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  10. Morett, E. & Segovia, L. ( 1993; ). The σ54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains. J Bacteriol 175, 6067-6074.
    [Google Scholar]
  11. North, A. K., Klose, K. E., Stedman, K. M. & Kustu, S. ( 1993; ). Prokaryotic enhancer-binding proteins reflect eukaryote-like modularity: puzzle of nitrogen regulatory protein C. J Bacteriol 175, 4267-4273.
    [Google Scholar]
  12. North, A. K., Weiss, D., Suzuky, H., Flashner, Y. & Kustu, S. ( 1996; ). Repressor forms of the enhancer-binding protein NtrC: some fail in coupling ATP hydrolysis to open complex formation by σ54-holoenzyme. J Mol Biol 260, 317-331.[CrossRef]
    [Google Scholar]
  13. Pérez-Martı́n, J. & de Lorenzo, V. ( 1995; ). The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor. Proc Natl Acad Sci U S A 92, 9392-9396.[CrossRef]
    [Google Scholar]
  14. Pérez-Martı́n, J. & de Lorenzo, V. ( 1996a; ). ATP binding to the σ54-dependent activator XylR triggers a protein multimerization cycle catalyzed by UAS DNA. Cell 86, 331-339.[CrossRef]
    [Google Scholar]
  15. Pérez-Martı́n, J. & de Lorenzo, V. ( 1996b; ). Physical and functional analysis of the prokaryotic enhancer of the σ54-promoters of the TOL plasmid of Pseudomonas putida. J Mol Biol 258, 562-574.[CrossRef]
    [Google Scholar]
  16. Pérez-Martı́n, J. & de Lorenzo, V. ( 1996c; ). In vitro activities of an N-terminal truncated form of XylR, a σ54-dependent transcriptional activator of Pseudomonas putida. J Mol Biol 258, 575-587.[CrossRef]
    [Google Scholar]
  17. Porter, S., North, A. K., Wedel, A. B. & Kustu, S. ( 1993; ). Oligomerization of NtrC at the glnA enhancer is required for transcriptional activation. Genes Dev 7, 2258-2273.[CrossRef]
    [Google Scholar]
  18. Révet, B., Brahms, S. & Brahms, G. ( 1995; ). Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation. Proc Natl Acad Sci U S A 92, 7535-7539.[CrossRef]
    [Google Scholar]
  19. Rippe, K., Guthold, M., Hippel, P. H. & Bustamante, C. ( 1997; ). Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase σ54 holoenzyme by scanning force microscopy. J Mol Biol 270, 125-138.[CrossRef]
    [Google Scholar]
  20. Rippe, K., Mücke, N. & Schulz, A. ( 1998; ). Association states of the transcription activator protein NtrC from E. coli determinated by analytical ultracentrifugation. J Mol Biol 278, 915-933.[CrossRef]
    [Google Scholar]
  21. Wyman, C., Rombel, I., North, A. K., Bustamante, C. & Kustu, S. ( 1997; ). Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. Science 275, 1658-1661.[CrossRef]
    [Google Scholar]
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