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. 1997 Aug 1;25(15):3042–3050. doi: 10.1093/nar/25.15.3042

NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein.

A L Vuidepot 1, F Bontems 1, M Gervais 1, B Guiard 1, E Shechter 1, J Y Lallemand 1
PMCID: PMC146857  PMID: 9224603

Abstract

The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure. The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography. Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element. They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity. Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion. To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B. Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream. The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues. This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.

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Selected References

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  1. Baleja J. D., Marmorstein R., Harrison S. C., Wagner G. Solution structure of the DNA-binding domain of Cd2-GAL4 from S. cerevisiae. Nature. 1992 Apr 2;356(6368):450–453. doi: 10.1038/356450a0. [DOI] [PubMed] [Google Scholar]
  2. Baleja J. D., Mau T., Wagner G. Recognition of DNA by GAL4 in solution: use of a monomeric protein-DNA complex for study by NMR. Biochemistry. 1994 Mar 15;33(10):3071–3078. doi: 10.1021/bi00176a041. [DOI] [PubMed] [Google Scholar]
  3. Clarke N. D., Beamer L. J., Goldberg H. R., Berkower C., Pabo C. O. The DNA binding arm of lambda repressor: critical contacts from a flexible region. Science. 1991 Oct 11;254(5029):267–270. doi: 10.1126/science.254.5029.267. [DOI] [PubMed] [Google Scholar]
  4. Creusot F., Verdière J., Gaisne M., Slonimski P. P. CYP1 (HAP1) regulator of oxygen-dependent gene expression in yeast. I. Overall organization of the protein sequence displays several novel structural domains. J Mol Biol. 1988 Nov 20;204(2):263–276. doi: 10.1016/0022-2836(88)90574-8. [DOI] [PubMed] [Google Scholar]
  5. Dhingra M. M., Sarma M. H., Gupta G., Sarma R. H. Determination of handedness of DNA double helices from NOE difference spectra: the structure of poly(dG-dC).poly(dG-dC) in low salt. J Biomol Struct Dyn. 1983 Oct;1(2):417–428. doi: 10.1080/07391102.1983.10507451. [DOI] [PubMed] [Google Scholar]
  6. Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [PubMed] [Google Scholar]
  7. Ha N., Hellauer K., Turcotte B. Mutations in target DNA elements of yeast HAP1 modulate its transcriptional activity without affecting DNA binding. Nucleic Acids Res. 1996 Apr 15;24(8):1453–1459. doi: 10.1093/nar/24.8.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  10. Liang S. D., Marmorstein R., Harrison S. C., Ptashne M. DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA. Mol Cell Biol. 1996 Jul;16(7):3773–3780. doi: 10.1128/mcb.16.7.3773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Marmorstein R., Carey M., Ptashne M., Harrison S. C. DNA recognition by GAL4: structure of a protein-DNA complex. Nature. 1992 Apr 2;356(6368):408–414. doi: 10.1038/356408a0. [DOI] [PubMed] [Google Scholar]
  12. Marmorstein R., Harrison S. C. Crystal structure of a PPR1-DNA complex: DNA recognition by proteins containing a Zn2Cys6 binuclear cluster. Genes Dev. 1994 Oct 15;8(20):2504–2512. doi: 10.1101/gad.8.20.2504. [DOI] [PubMed] [Google Scholar]
  13. Näit-Kaoudjt R., Williams R., Guiard B., Gervais M. Some DNA targets of the yeast CYP1 transcriptional activator are functionally asymmetric--evidence of two half-sites with different affinities. Eur J Biochem. 1997 Mar 1;244(2):301–309. doi: 10.1111/j.1432-1033.1997.00301.x. [DOI] [PubMed] [Google Scholar]
  14. Omichinski J. G., Pedone P. V., Felsenfeld G., Gronenborn A. M., Clore G. M. The solution structure of a specific GAGA factor-DNA complex reveals a modular binding mode. Nat Struct Biol. 1997 Feb;4(2):122–132. doi: 10.1038/nsb0297-122. [DOI] [PubMed] [Google Scholar]
  15. Pfeifer K., Kim K. S., Kogan S., Guarente L. Functional dissection and sequence of yeast HAP1 activator. Cell. 1989 Jan 27;56(2):291–301. doi: 10.1016/0092-8674(89)90903-3. [DOI] [PubMed] [Google Scholar]
  16. Pfeifer K., Prezant T., Guarente L. Yeast HAP1 activator binds to two upstream activation sites of different sequence. Cell. 1987 Apr 10;49(1):19–27. doi: 10.1016/0092-8674(87)90751-3. [DOI] [PubMed] [Google Scholar]
  17. Piotto M., Saudek V., Sklenár V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR. 1992 Nov;2(6):661–665. doi: 10.1007/BF02192855. [DOI] [PubMed] [Google Scholar]
  18. Reece R. J., Ptashne M. Determinants of binding-site specificity among yeast C6 zinc cluster proteins. Science. 1993 Aug 13;261(5123):909–911. doi: 10.1126/science.8346441. [DOI] [PubMed] [Google Scholar]
  19. Roy A., Exinger F., Losson R. cis- and trans-acting regulatory elements of the yeast URA3 promoter. Mol Cell Biol. 1990 Oct;10(10):5257–5270. doi: 10.1128/mcb.10.10.5257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schjerling P., Holmberg S. Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators. Nucleic Acids Res. 1996 Dec 1;24(23):4599–4607. doi: 10.1093/nar/24.23.4599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Szewczak A. A., Kellogg G. W., Moore P. B. Assignment of NH resonances in nucleic acids using natural abundance 15N-1H correlation spectroscopy with spin-echo and gradient pulses. FEBS Lett. 1993 Aug 2;327(3):261–264. doi: 10.1016/0014-5793(93)81000-p. [DOI] [PubMed] [Google Scholar]
  22. Timmerman J. E., Guiard B., Shechter E., Delsuc M. A., Lallemand J. Y., Gervais M. The DNA-binding domain of the yeast Saccharomyces cerevisiae CYP1(HAP1) transcription factor possesses two zinc ions which are complexed in a zinc cluster. Eur J Biochem. 1994 Oct 15;225(2):593–599. doi: 10.1111/j.1432-1033.1994.00593.x. [DOI] [PubMed] [Google Scholar]
  23. Timmerman J., Vuidepot A. L., Bontems F., Lallemand J. Y., Gervais M., Shechter E., Guiard B. 1H, 15N resonance assignment and three-dimensional structure of CYP1 (HAP1) DNA-binding domain. J Mol Biol. 1996 Jun 21;259(4):792–804. doi: 10.1006/jmbi.1996.0358. [DOI] [PubMed] [Google Scholar]
  24. Turcotte B., Guarente L. HAP1 positive control mutants specific for one of two binding sites. Genes Dev. 1992 Oct;6(10):2001–2009. doi: 10.1101/gad.6.10.2001. [DOI] [PubMed] [Google Scholar]
  25. Verdière J., Creusot F., Guérineau M. Regulation of the expression of iso 2-cytochrome c gene in S. cerevisiae: cloning of the positive regulatory gene CYP1 and identification of the region of its target sequence on the structural gene CYP3. Mol Gen Genet. 1985;199(3):524–533. doi: 10.1007/BF00330769. [DOI] [PubMed] [Google Scholar]
  26. Zhang L., Guarente L. The yeast activator HAP1--a GAL4 family member--binds DNA in a directly repeated orientation. Genes Dev. 1994 Sep 1;8(17):2110–2119. doi: 10.1101/gad.8.17.2110. [DOI] [PubMed] [Google Scholar]

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