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. 1993 Oct 1;90(19):9186–9190. doi: 10.1073/pnas.90.19.9186

A genetic method for defining DNA-binding domains: application to the nuclear receptor NGFI-B.

T E Wilson 1, K A Padgett 1, M Johnston 1, J Milbrandt 1
PMCID: PMC47527  PMID: 8415675

Abstract

A method is described that allows for rapid and efficient generation of functional mutations in DNA-binding domains of proteins. The target DNA-binding domain is attached to the Gal4p transcriptional-activating domain and expressed in yeast. The binding site recognized by the target domain is placed upstream of a gene that produces a protein toxic to yeast cells, so that the chimeric protein activates its expression, providing a selection against DNA-binding domain function. The chimeric protein also activates expression of a gene necessary for histidine prototrophy, using a second DNA-binding domain included in the chimera (lexA), providing a selection against general activator mutations. Therefore, requiring growth in the absence of histidine focuses mutations to the target DNA-binding domain. This method was applied to the DNA-binding domain of the nuclear receptor NGFI-B. Nearly all mutations obtained concurred with previous studies of NGFI-B and other nuclear receptors, verifying the functional validity of the mutational profile obtained. In addition, by coupling this selection scheme with the two-hybrid system [Chien, C.-t., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) Proc. Natl. Acad. Sci. USA 88, 9578-9582], mutations that alter protein interaction domains could also be obtained.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  2. Berg J. M. DNA binding specificity of steroid receptors. Cell. 1989 Jun 30;57(7):1065–1068. doi: 10.1016/0092-8674(89)90042-1. [DOI] [PubMed] [Google Scholar]
  3. Berg J. M. Zinc finger domains: hypotheses and current knowledge. Annu Rev Biophys Biophys Chem. 1990;19:405–421. doi: 10.1146/annurev.bb.19.060190.002201. [DOI] [PubMed] [Google Scholar]
  4. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  5. Brent R., Ptashne M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell. 1985 Dec;43(3 Pt 2):729–736. doi: 10.1016/0092-8674(85)90246-6. [DOI] [PubMed] [Google Scholar]
  6. Busch S. J., Sassone-Corsi P. Dimers, leucine zippers and DNA-binding domains. Trends Genet. 1990 Feb;6(2):36–40. doi: 10.1016/0168-9525(90)90071-d. [DOI] [PubMed] [Google Scholar]
  7. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  8. Callahan A. M., Frazier B. L., Parkinson J. S. Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage. J Bacteriol. 1987 Mar;169(3):1246–1253. doi: 10.1128/jb.169.3.1246-1253.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DOUGLAS H. C., HAWTHORNE D. C. ENZYMATIC EXPRESSION AND GENETIC LINKAGE OF GENES CONTROLLING GALACTOSE UTILIZATION IN SACCHAROMYCES. Genetics. 1964 May;49:837–844. doi: 10.1093/genetics/49.5.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dynan W. S., Tjian R. Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. 1985 Aug 29-Sep 4Nature. 316(6031):774–778. doi: 10.1038/316774a0. [DOI] [PubMed] [Google Scholar]
  12. Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  14. Giguere V., Ong E. S., Segui P., Evans R. M. Identification of a receptor for the morphogen retinoic acid. Nature. 1987 Dec 17;330(6149):624–629. doi: 10.1038/330624a0. [DOI] [PubMed] [Google Scholar]
  15. Green S., Walter P., Kumar V., Krust A., Bornert J. M., Argos P., Chambon P. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 1986 Mar 13;320(6058):134–139. doi: 10.1038/320134a0. [DOI] [PubMed] [Google Scholar]
  16. Gronemeyer H., Turcotte B., Quirin-Stricker C., Bocquel M. T., Meyer M. E., Krozowski Z., Jeltsch J. M., Lerouge T., Garnier J. M., Chambon P. The chicken progesterone receptor: sequence, expression and functional analysis. EMBO J. 1987 Dec 20;6(13):3985–3994. doi: 10.1002/j.1460-2075.1987.tb02741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harrison S. C. A structural taxonomy of DNA-binding domains. Nature. 1991 Oct 24;353(6346):715–719. doi: 10.1038/353715a0. [DOI] [PubMed] [Google Scholar]
  18. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  19. Heyman R. A., Mangelsdorf D. J., Dyck J. A., Stein R. B., Eichele G., Evans R. M., Thaller C. 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992 Jan 24;68(2):397–406. doi: 10.1016/0092-8674(92)90479-v. [DOI] [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnson P. F., McKnight S. L. Eukaryotic transcriptional regulatory proteins. Annu Rev Biochem. 1989;58:799–839. doi: 10.1146/annurev.bi.58.070189.004055. [DOI] [PubMed] [Google Scholar]
  22. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lala D. S., Rice D. A., Parker K. L. Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol Endocrinol. 1992 Aug;6(8):1249–1258. doi: 10.1210/mend.6.8.1406703. [DOI] [PubMed] [Google Scholar]
  24. Luisi B. F., Xu W. X., Otwinowski Z., Freedman L. P., Yamamoto K. R., Sigler P. B. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991 Aug 8;352(6335):497–505. doi: 10.1038/352497a0. [DOI] [PubMed] [Google Scholar]
  25. Ma J., Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987 Mar 13;48(5):847–853. doi: 10.1016/0092-8674(87)90081-x. [DOI] [PubMed] [Google Scholar]
  26. Milbrandt J. Nerve growth factor induces a gene homologous to the glucocorticoid receptor gene. Neuron. 1988 May;1(3):183–188. doi: 10.1016/0896-6273(88)90138-9. [DOI] [PubMed] [Google Scholar]
  27. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  28. När A. M., Boutin J. M., Lipkin S. M., Yu V. C., Holloway J. M., Glass C. K., Rosenfeld M. G. The orientation and spacing of core DNA-binding motifs dictate selective transcriptional responses to three nuclear receptors. Cell. 1991 Jun 28;65(7):1267–1279. doi: 10.1016/0092-8674(91)90021-p. [DOI] [PubMed] [Google Scholar]
  29. Oertel-Buchheit P., Schmidt-Dörr T., Granger-Schnarr M., Schnarr M. Spacing requirements between LexA operator half-sites can be relaxed by fusing the LexA DNA binding domain with some alternative dimerization domains. J Mol Biol. 1993 Jan 5;229(1):1–7. doi: 10.1006/jmbi.1993.1001. [DOI] [PubMed] [Google Scholar]
  30. Pavletich N. P., Pabo C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science. 1991 May 10;252(5007):809–817. doi: 10.1126/science.2028256. [DOI] [PubMed] [Google Scholar]
  31. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  32. Schaaper R. M., Radman M. The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors. EMBO J. 1989 Nov;8(11):3511–3516. doi: 10.1002/j.1460-2075.1989.tb08516.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schena M., Freedman L. P., Yamamoto K. R. Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities. Genes Dev. 1989 Oct;3(10):1590–1601. doi: 10.1101/gad.3.10.1590. [DOI] [PubMed] [Google Scholar]
  34. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schwabe J. W., Neuhaus D., Rhodes D. Solution structure of the DNA-binding domain of the oestrogen receptor. Nature. 1990 Nov 29;348(6300):458–461. doi: 10.1038/348458a0. [DOI] [PubMed] [Google Scholar]
  36. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  37. Tan J. A., Joseph D. R., Quarmby V. E., Lubahn D. B., Sar M., French F. S., Wilson E. M. The rat androgen receptor: primary structure, autoregulation of its messenger ribonucleic acid, and immunocytochemical localization of the receptor protein. Mol Endocrinol. 1988 Dec;2(12):1276–1285. doi: 10.1210/mend-2-12-1276. [DOI] [PubMed] [Google Scholar]
  38. Umesono K., Evans R. M. Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell. 1989 Jun 30;57(7):1139–1146. doi: 10.1016/0092-8674(89)90051-2. [DOI] [PubMed] [Google Scholar]
  39. Umesono K., Murakami K. K., Thompson C. C., Evans R. M. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell. 1991 Jun 28;65(7):1255–1266. doi: 10.1016/0092-8674(91)90020-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
  41. Wang L. H., Tsai S. Y., Cook R. G., Beattie W. G., Tsai M. J., O'Malley B. W. COUP transcription factor is a member of the steroid receptor superfamily. Nature. 1989 Jul 13;340(6229):163–166. doi: 10.1038/340163a0. [DOI] [PubMed] [Google Scholar]
  42. Weinberger C., Hollenberg S. M., Ong E. S., Harmon J. M., Brower S. T., Cidlowski J., Thompson E. B., Rosenfeld M. G., Evans R. M. Identification of human glucocorticoid receptor complementary DNA clones by epitope selection. Science. 1985 May 10;228(4700):740–742. doi: 10.1126/science.2581314. [DOI] [PubMed] [Google Scholar]
  43. Weinberger C., Thompson C. C., Ong E. S., Lebo R., Gruol D. J., Evans R. M. The c-erb-A gene encodes a thyroid hormone receptor. Nature. 1986 Dec 18;324(6098):641–646. doi: 10.1038/324641a0. [DOI] [PubMed] [Google Scholar]
  44. Wilson T. E., Day M. L., Pexton T., Padgett K. A., Johnston M., Milbrandt J. In vivo mutational analysis of the NGFI-A zinc fingers. J Biol Chem. 1992 Feb 25;267(6):3718–3724. [PubMed] [Google Scholar]
  45. Wilson T. E., Fahrner T. J., Johnston M., Milbrandt J. Identification of the DNA binding site for NGFI-B by genetic selection in yeast. Science. 1991 May 31;252(5010):1296–1300. doi: 10.1126/science.1925541. [DOI] [PubMed] [Google Scholar]
  46. Wilson T. E., Mouw A. R., Weaver C. A., Milbrandt J., Parker K. L. The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase. Mol Cell Biol. 1993 Feb;13(2):861–868. doi: 10.1128/mcb.13.2.861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wilson T. E., Paulsen R. E., Padgett K. A., Milbrandt J. Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors. Science. 1992 Apr 3;256(5053):107–110. doi: 10.1126/science.1314418. [DOI] [PubMed] [Google Scholar]
  48. Wolberger C., Vershon A. K., Liu B., Johnson A. D., Pabo C. O. Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions. Cell. 1991 Nov 1;67(3):517–528. doi: 10.1016/0092-8674(91)90526-5. [DOI] [PubMed] [Google Scholar]

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