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Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA

Nature Structural Biology volume 7, pages 424–430 (2000)Cite this article

Abstract

The Escherichia coli Rob protein is a transcription factor belonging to the AraC/XylS protein family that regulates genes involved in resistance to antibiotics, organic solvents and heavy metals. The genes encoding these proteins are activated by the homologous proteins MarA and SoxS, although the level of activation can vary for the different transcription factors. Here we report a 2.7 Å crystal structure of Rob in complex with the micF promoter that reveals an unusual mode of binding to DNA. The Rob–DNA complex differs from the previously reported structure of MarA bound to the mar promoter, in that only one of Rob's dual helix-turn-helix (HTH) motifs engages the major groove of the binding site. Biochemical studies show that sequence specific interactions involving only one of Rob's HTH motifs are sufficient for high affinity binding to DNA. The two different modes of DNA binding seen in crystal structures of Rob and MarA also match the distinctive patterns of DNA protection by AraC at several sites within the pBAD promoter. These and other findings suggest that gene activation by AraC/XylS transcription factors might involve two alternative modes of binding to DNA in different promoter contexts.

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Figure 1: The structure of Rob in complex with the micF promotor.
Figure 2: Interactions of Rob with DNA.
Figure 3: Sequence alignment of AraC/XylS family members.
Figure 4: Rob's C-terminal domain might restrict DNA bending.
Figure 5: DNA binding affinities for modified promoter sequences.
Figure 6: Structural similarity of Rob and GalT.
Figure 7: A model of AraC in complex with the pBAD promoter.

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References

  1. Skarstad, K., Thony, B., Hwang, D.S. & Kornberg, A. A novel binding protein of the origin of the Escherichia coli chromosome. J. Biol. Chem. 268, 5365–5370 (1993).

    CAS  PubMed  Google Scholar 

  2. Gallegos, M.T., Schleif, R., Bairoch, A., Hofmann, K. & Ramos, J.L. Arac/XylS family of transcriptional regulators. Microbiol. Mol. Biol. Rev. 61, 393–410 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Demple, B. Redox signaling and gene control in the Escherichia coli soxRS oxidative stress regulon — a review. Gene 179, 53–57 (1996).

    Article  CAS  Google Scholar 

  4. Alekshun, M.N. & Levy, S.B. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob. Agents. Chemother. 41, 2067–2075 (1997).

    Article  CAS  Google Scholar 

  5. Cohen, S.P., Hachler, H. & Levy, S.B. Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli. J. Bacteriol. 175, 1484–1492 (1993).

    Article  CAS  Google Scholar 

  6. Ariza, R.R., Li, Z., Ringstad, N. & Demple, B. Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein. J. Bacteriol. 177, 1655–1661 (1995).

    Article  CAS  Google Scholar 

  7. Nakajima, H., Kobayashi, K., Kobayashi, M., Asako, H. & Aono, R. Overexpression of the robA gene increases organic solvent tolerance and multiple antibiotic and heavy metal ion resistance in Escherichia coli. Appl. Environ. Microbiol. 61, 2302–2307 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. White, D.G., Goldman, J.D., Demple, B. & Levy, S.B. Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli. J. Bacteriol. 179, 6122–6126 (1997).

    Article  CAS  Google Scholar 

  9. Jair, K.W. et al. Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication. J. Bacteriol. 178, 2507–2513 (1996).

    Article  CAS  Google Scholar 

  10. Greenberg, J.T., Chou, J.H., Monach, P.A. & Demple, B. Activation of oxidative stress genes by mutations at the soxQ/cfxB/marA locus of Escherichia coli. J. Bacteriol. 173, 4433–4439 (1991).

    Article  CAS  Google Scholar 

  11. Ariza, R.R., Cohen, S.P., Bachhawat, N., Levy, S.B. & Demple, B. Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli. J. Bacteriol. 176, 143–148 (1994).

    Article  CAS  Google Scholar 

  12. Martin, R.G., Gillette, W.K., Rhee, S. & Rosner, J.L. Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spatial relationship to the core promoter [In Process Citation]. Mol. Microbiol. 34, 431–441 (1999).

    Article  CAS  Google Scholar 

  13. Rhee, S., Martin, R.G., Rosner, J.L. & Davies, D.R. A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator. Proc. Natl Acad. Sci. USA 95, 10413–10418 (1998).

    Article  CAS  Google Scholar 

  14. Kakeda, M., Ueguchi, C., Yamada, H. & Mizuno, T. An Escherichia coli curved DNA-binding protein whose expression is affected by the stationary phase-specific sigma factor sigma S. Mol. Gen. Genet. 248, 629–634 (1995).

    Article  CAS  Google Scholar 

  15. Lobell, R.B. & Schleif, R.F. DNA looping and unlooping by AraC protein. Science 250, 528–532 (1990).

    Article  CAS  Google Scholar 

  16. Ramos, J.L., Michan, C., Rojo, F., Dwyer, D. & Timmis, K. Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon. J. Mol. Biol. 211, 373–382 (1990).

    Article  CAS  Google Scholar 

  17. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  18. Thoden, J.B., Ruzicka, F.J., Frey, P.A., Rayment, I. & Holden, H.M. Structural analysis of the H166G site-directed mutant of galactose-1-phosphate uridylyltransferase complexed with either UDP-glucose or UDP-galactose: detailed description of the nucleotide sugar binding site. Biochemistry 36, 1212–1222 (1997).

    Article  CAS  Google Scholar 

  19. Li, Z. & Demple, B. SoxS, an activator of superoxide stress genes in Escherichia coli. Purification and interaction with DNA. J. Biol. Chem. 269, 18371–18377 (1994).

    CAS  PubMed  Google Scholar 

  20. Zhang, X., Reeder, T. & Schleif, R. Transcription activation parameters at ara pBAD. J. Mol. Biol. 258, 14–24 (1996).

    Article  CAS  Google Scholar 

  21. Jair, K.W., Fawcett, W.P., Fujita, N., Ishihama, A., Wolf, R.E. Jr., Ambidextrous transcriptional activation by SoxS: requirement for the C-terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide-inducible genes. Mol. Microbiol. 19, 307–317 (1996).

    Article  CAS  Google Scholar 

  22. Li, Z. & Demple, B. Sequence specificity for DNA binding by Escherichia coli SoxS and Rob proteins. Mol. Microbiol. 20, 937–945 (1996).

    Article  CAS  Google Scholar 

  23. Martin, R.G., Gillette, W.K., Rhee, S. & Rosner, J.L. Promoter discrimination by the related transcriptional activators MarA and SoxS: differential regulation by differential binding. Molec. Microbiol. 35, 623–634 (2000).

    Article  CAS  Google Scholar 

  24. Niland, P., Huhne, R. & Muller-Hill, B. How AraC interacts specifically with its target DNAs. J. Mol. Biol. 264, 667–674 (1996).

    Article  CAS  Google Scholar 

  25. Reeder, T. & Schleif, R. AraC protein can activate transcription from only one position and when pointed in only one direction. J. Mol. Biol. 231, 205–218 (1993).

    Article  CAS  Google Scholar 

  26. Brunelle, A. & Schleif, R. Determining residue-base interactions between AraC protein and araI DNA. J. Mol. Biol. 209, 607–622 (1989).

    Article  CAS  Google Scholar 

  27. Hendrickson, W. & Schleif, R. A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site. Proc. Natl Acad. Sci. USA 82, 3129–3133 (1985).

    Article  CAS  Google Scholar 

  28. Martin, R.G., Jair, K.W., Wolf, R.E. Jr. & Rosner, J.L. Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli. J. Bacteriol. 178, 2216–2223 (1996).

    Article  CAS  Google Scholar 

  29. Koh, Y.S., Chung, W.-H., Lee, J.-H. & Roe, J.-H. The reversed SoxS-binding site upstream of the ribA promoter in Escherichia coli. Mol. Gen. Genet. 261, 374–380 (1999).

    Article  CAS  Google Scholar 

  30. Nunoshiba, T., Hidalgo, E., Li, Z. & Demple, B. Negative autoregulation by the Escherichia coli SoxS protein: a dampening mechanism for the soxRS redox stress response. J. Bacteriol. 175, 7492–7494 (1993).

    Article  CAS  Google Scholar 

  31. Wood, T.I. et al. Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters. Mol. Microbiol. 34, 414–430 (1999).

    Article  CAS  Google Scholar 

  32. Doublie, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).

    Article  CAS  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 461–472 (1997).

    Article  Google Scholar 

  34. Bailey, S. The CCP4 suite: programs for protein crystallography. Acta Crystallog. D 52, 30–42 (1994).

    Google Scholar 

  35. Abrahams, J.P. & Leslie, A.G.W. Methods used in the structure determination of bovine mitochrondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996).

    Article  CAS  Google Scholar 

  36. Brunger, A.T. et al. Crystallography,NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  37. Jones, T.A. O: the manual (, Uppsala, Sweden; 1992) http://kaktus.kemi.aau.dk

  38. Brunger, A.T. The free R-value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–474 (1992).

    Article  CAS  Google Scholar 

  39. Evans, S.V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J. Mol. Graph. 11, 134–138, (1993).

    Article  CAS  Google Scholar 

  40. Barton, G.J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37–40 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Lau, M. Sawaya and members of the Hogle research group for assistance with X-ray data collection. We appreciate the generous gift of purified MarA protein from M. Alekshun and S. Levy (Tufts University School of Medicine). This work was supported by grants from the National Institutes of Health (T.E, B.D.), a Howard Hughes Medical Institute Predoctoral Fellowship (H.J.K.), and the Agrotechnological Research Institute, The Netherlands (M.H.J.B.). We also acknowledge the support of the Giovanni Armenise Foundation for Advanced Scientific Research and the Harvard Center for Structural Biology.

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  1. Marjon H.J. Bennik

    Present address: Agrotechnological Research Institute, Wageningen University Research Centre, 6700 AA, Wageningen, The Netherlands

Authors and Affiliations

  1. Graduate Program in Biophysics, Harvard University, Cambridge, 02138, MA, USA

    Hyock Joo Kwon & Tom Ellenberger

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, 02115, MA, USA

    Hyock Joo Kwon & Tom Ellenberger

  3. Department of Cancer Cell Biology, Harvard School of Public Health, Boston, 02115, MA, USA

    Marjon H.J. Bennik & Bruce Demple

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Correspondence to Tom Ellenberger.

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Kwon, H., Bennik, M., Demple, B. et al. Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA. Nat Struct Mol Biol 7, 424–430 (2000). https://doi.org/10.1038/75213

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