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. 1996 Nov 15;24(22):4487–4494. doi: 10.1093/nar/24.22.4487

DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

M John 1, R Leppik 1, S J Busch 1, M Granger-Schnarr 1, M Schnarr 1
PMCID: PMC146289  PMID: 8948639

Abstract

We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.

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Selected References

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  1. Abate C., Luk D., Gentz R., Rauscher F. J., 3rd, Curran T. Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1032–1036. doi: 10.1073/pnas.87.3.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angel P., Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):129–157. doi: 10.1016/0304-419x(91)90011-9. [DOI] [PubMed] [Google Scholar]
  3. Brent R., Ptashne M. Mechanism of action of the lexA gene product. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4204–4208. doi: 10.1073/pnas.78.7.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [DOI] [PubMed] [Google Scholar]
  5. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  6. Glover J. N., Harrison S. C. Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature. 1995 Jan 19;373(6511):257–261. doi: 10.1038/373257a0. [DOI] [PubMed] [Google Scholar]
  7. Hurst H. C. Transcription factors. 1: bZIP proteins. Protein Profile. 1994;1(2):123–168. [PubMed] [Google Scholar]
  8. Ivanov V. I., Krylov DYu A-DNA in solution as studied by diverse approaches. Methods Enzymol. 1992;211:111–127. doi: 10.1016/0076-6879(92)11008-7. [DOI] [PubMed] [Google Scholar]
  9. Keller W., König P., Richmond T. J. Crystal structure of a bZIP/DNA complex at 2.2 A: determinants of DNA specific recognition. J Mol Biol. 1995 Dec 8;254(4):657–667. doi: 10.1006/jmbi.1995.0645. [DOI] [PubMed] [Google Scholar]
  10. Kerppola T. K., Curran T. DNA bending by Fos and Jun: the flexible hinge model. Science. 1991 Nov 22;254(5035):1210–1214. doi: 10.1126/science.1957173. [DOI] [PubMed] [Google Scholar]
  11. Kerppola T. K., Curran T. Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity. Cell. 1991 Jul 26;66(2):317–326. doi: 10.1016/0092-8674(91)90621-5. [DOI] [PubMed] [Google Scholar]
  12. Kerppola T. K., Curran T. Selective DNA bending by a variety of bZIP proteins. Mol Cell Biol. 1993 Sep;13(9):5479–5489. doi: 10.1128/mcb.13.9.5479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. König P., Richmond T. J. The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA flexibility. J Mol Biol. 1993 Sep 5;233(1):139–154. doi: 10.1006/jmbi.1993.1490. [DOI] [PubMed] [Google Scholar]
  14. Nakabeppu Y., Nathans D. The basic region of Fos mediates specific DNA binding. EMBO J. 1989 Dec 1;8(12):3833–3841. doi: 10.1002/j.1460-2075.1989.tb08561.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nekludova L., Pabo C. O. Distinctive DNA conformation with enlarged major groove is found in Zn-finger-DNA and other protein-DNA complexes. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6948–6952. doi: 10.1073/pnas.91.15.6948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Patel L. R., Curran T., Kerppola T. K. Energy transfer analysis of Fos-Jun dimerization and DNA binding. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7360–7364. doi: 10.1073/pnas.91.15.7360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Patel L., Abate C., Curran T. Altered protein conformation on DNA binding by Fos and Jun. Nature. 1990 Oct 11;347(6293):572–575. doi: 10.1038/347572a0. [DOI] [PubMed] [Google Scholar]
  18. Ryseck R. P., Bravo R. c-JUN, JUN B, and JUN D differ in their binding affinities to AP-1 and CRE consensus sequences: effect of FOS proteins. Oncogene. 1991 Apr;6(4):533–542. [PubMed] [Google Scholar]
  19. Schmidt-Dörr T., Oertel-Buchheit P., Pernelle C., Bracco L., Schnarr M., Granger-Schnarr M. Construction, purification, and characterization of a hybrid protein comprising the DNA binding domain of the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study. Biochemistry. 1991 Oct 8;30(40):9657–9664. doi: 10.1021/bi00104a013. [DOI] [PubMed] [Google Scholar]
  20. Scopes R. K. Measurement of protein by spectrophotometry at 205 nm. Anal Biochem. 1974 May;59(1):277–282. doi: 10.1016/0003-2697(74)90034-7. [DOI] [PubMed] [Google Scholar]
  21. Sitlani A., Crothers D. M. Fos and Jun do not bend the AP-1 recognition site. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3248–3252. doi: 10.1073/pnas.93.8.3248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sprous D., Zacharias W., Wood Z. A., Harvey S. C. Dehydrating agents sharply reduce curvature in DNAs containing A tracts. Nucleic Acids Res. 1995 May 25;23(10):1816–1821. doi: 10.1093/nar/23.10.1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Weiss M. A., Ellenberger T., Wobbe C. R., Lee J. P., Harrison S. C., Struhl K. Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA. Nature. 1990 Oct 11;347(6293):575–578. doi: 10.1038/347575a0. [DOI] [PubMed] [Google Scholar]
  24. Woody R. W. Circular dichroism. Methods Enzymol. 1995;246:34–71. doi: 10.1016/0076-6879(95)46006-3. [DOI] [PubMed] [Google Scholar]
  25. Xanthoudakis S., Curran T. Analysis of c-Fos and c-Jun redox-dependent DNA binding activity. Methods Enzymol. 1994;234:163–174. doi: 10.1016/0076-6879(94)34086-2. [DOI] [PubMed] [Google Scholar]
  26. Zhong L., Johnson W. C., Jr Environment affects amino acid preference for secondary structure. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4462–4465. doi: 10.1073/pnas.89.10.4462. [DOI] [PMC free article] [PubMed] [Google Scholar]

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