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. 1990 Jun;10(6):2916–2923. doi: 10.1128/mcb.10.6.2916

GAL4 protein: purification, association with GAL80 protein, and conserved domain structure.

D I Chasman 1, R D Kornberg 1
PMCID: PMC360654  PMID: 2188103

Abstract

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

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Selected References

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  1. Bajwa W., Torchia T. E., Hopper J. E. Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases. Mol Cell Biol. 1988 Aug;8(8):3439–3447. doi: 10.1128/mcb.8.8.3439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balzi E., Chen W., Ulaszewski S., Capieaux E., Goffeau A. The multidrug resistance gene PDR1 from Saccharomyces cerevisiae. J Biol Chem. 1987 Dec 15;262(35):16871–16879. [PubMed] [Google Scholar]
  3. Beri R. K., Whittington H., Roberts C. F., Hawkins A. R. Isolation and characterization of the positively acting regulatory gene QUTA from Aspergillus nidulans. Nucleic Acids Res. 1987 Oct 12;15(19):7991–8001. doi: 10.1093/nar/15.19.7991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Bram R. J., Kornberg R. D. Specific protein binding to far upstream activating sequences in polymerase II promoters. Proc Natl Acad Sci U S A. 1985 Jan;82(1):43–47. doi: 10.1073/pnas.82.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buchman A. R., Kimmerly W. J., Rine J., Kornberg R. D. Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jan;8(1):210–225. doi: 10.1128/mcb.8.1.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buchman A. R., Lue N. F., Kornberg R. D. Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein. Mol Cell Biol. 1988 Dec;8(12):5086–5099. doi: 10.1128/mcb.8.12.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buratowski S., Hahn S., Guarente L., Sharp P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. doi: 10.1016/0092-8674(89)90578-3. [DOI] [PubMed] [Google Scholar]
  9. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  10. Bushman F. D., Shang C., Ptashne M. A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor. Cell. 1989 Sep 22;58(6):1163–1171. doi: 10.1016/0092-8674(89)90514-x. [DOI] [PubMed] [Google Scholar]
  11. Carey M., Kakidani H., Leatherwood J., Mostashari F., Ptashne M. An amino-terminal fragment of GAL4 binds DNA as a dimer. J Mol Biol. 1989 Oct 5;209(3):423–432. doi: 10.1016/0022-2836(89)90007-7. [DOI] [PubMed] [Google Scholar]
  12. Chasman D. I., Leatherwood J., Carey M., Ptashne M., Kornberg R. D. Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro. Mol Cell Biol. 1989 Nov;9(11):4746–4749. doi: 10.1128/mcb.9.11.4746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chasman D. I., Lue N. F., Buchman A. R., LaPointe J. W., Lorch Y., Kornberg R. D. A yeast protein that influences the chromatin structure of UASG and functions as a powerful auxiliary gene activator. Genes Dev. 1990 Apr;4(4):503–514. doi: 10.1101/gad.4.4.503. [DOI] [PubMed] [Google Scholar]
  14. Corton J. C., Johnston S. A. Altering DNA-binding specificity of GAL4 requires sequences adjacent to the zinc finger. Nature. 1989 Aug 31;340(6236):724–727. doi: 10.1038/340724a0. [DOI] [PubMed] [Google Scholar]
  15. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Evans R. M., Hollenberg S. M. Zinc fingers: gilt by association. Cell. 1988 Jan 15;52(1):1–3. doi: 10.1016/0092-8674(88)90522-3. [DOI] [PubMed] [Google Scholar]
  17. Fedor M. J., Lue N. F., Kornberg R. D. Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence in yeast. J Mol Biol. 1988 Nov 5;204(1):109–127. doi: 10.1016/0022-2836(88)90603-1. [DOI] [PubMed] [Google Scholar]
  18. Friden P., Reynolds C., Schimmel P. A large internal deletion converts yeast LEU3 to a constitutive transcriptional activator. Mol Cell Biol. 1989 Sep;9(9):4056–4060. doi: 10.1128/mcb.9.9.4056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Friden P., Schimmel P. LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes. Mol Cell Biol. 1987 Aug;7(8):2708–2717. doi: 10.1128/mcb.7.8.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Jackson S. P., Tjian R. O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell. 1988 Oct 7;55(1):125–133. doi: 10.1016/0092-8674(88)90015-3. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Johnston M., Dover J. Mutational analysis of the GAL4-encoded transcriptional activator protein of Saccharomyces cerevisiae. Genetics. 1988 Sep;120(1):63–74. doi: 10.1093/genetics/120.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johnston M. Genetic evidence that zinc is an essential co-factor in the DNA binding domain of GAL4 protein. Nature. 1987 Jul 23;328(6128):353–355. doi: 10.1038/328353a0. [DOI] [PubMed] [Google Scholar]
  28. Johnston S. A., Hopper J. E. Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6971–6975. doi: 10.1073/pnas.79.22.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Johnston S. A., Salmeron J. M., Jr, Dincher S. S. Interaction of positive and negative regulatory proteins in the galactose regulon of yeast. Cell. 1987 Jul 3;50(1):143–146. doi: 10.1016/0092-8674(87)90671-4. [DOI] [PubMed] [Google Scholar]
  30. Johnston S. A., Zavortink M. J., Debouck C., Hopper J. E. Functional domains of the yeast regulatory protein GAL4. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6553–6557. doi: 10.1073/pnas.83.17.6553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kakidani H., Ptashne M. GAL4 activates gene expression in mammalian cells. Cell. 1988 Jan 29;52(2):161–167. doi: 10.1016/0092-8674(88)90504-1. [DOI] [PubMed] [Google Scholar]
  33. Kammerer B., Guyonvarch A., Hubert J. C. Yeast regulatory gene PPR1. I. Nucleotide sequence, restriction map and codon usage. J Mol Biol. 1984 Dec 5;180(2):239–250. doi: 10.1016/s0022-2836(84)80002-9. [DOI] [PubMed] [Google Scholar]
  34. Laughon A., Gesteland R. F. Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol. 1984 Feb;4(2):260–267. doi: 10.1128/mcb.4.2.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lin Y. S., Carey M. F., Ptashne M., Green M. R. GAL4 derivatives function alone and synergistically with mammalian activators in vitro. Cell. 1988 Aug 26;54(5):659–664. doi: 10.1016/s0092-8674(88)80010-2. [DOI] [PubMed] [Google Scholar]
  36. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  37. Lorch Y., Kornberg R. D. A region flanking the GAL7 gene and a binding site for GAL4 protein as upstream activating sequences in yeast. J Mol Biol. 1985 Dec 20;186(4):821–824. doi: 10.1016/0022-2836(85)90400-0. [DOI] [PubMed] [Google Scholar]
  38. Lue N. F., Chasman D. I., Buchman A. R., Kornberg R. D. Interaction of GAL4 and GAL80 gene regulatory proteins in vitro. Mol Cell Biol. 1987 Oct;7(10):3446–3451. doi: 10.1128/mcb.7.10.3446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Ma J., Ptashne M. The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80. Cell. 1987 Jul 3;50(1):137–142. doi: 10.1016/0092-8674(87)90670-2. [DOI] [PubMed] [Google Scholar]
  41. McLachlan A. D. Tests for comparing related amino-acid sequences. Cytochrome c and cytochrome c 551 . J Mol Biol. 1971 Oct 28;61(2):409–424. doi: 10.1016/0022-2836(71)90390-1. [DOI] [PubMed] [Google Scholar]
  42. Mylin L. M., Bhat J. P., Hopper J. E. Regulated phosphorylation and dephosphorylation of GAL4, a transcriptional activator. Genes Dev. 1989 Aug;3(8):1157–1165. doi: 10.1101/gad.3.8.1157. [DOI] [PubMed] [Google Scholar]
  43. Nogi Y., Fukasawa T. Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jul;9(7):3009–3017. doi: 10.1128/mcb.9.7.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nogi Y., Fukasawa T. Nucleotide sequence of the yeast regulatory gene GAL80. Nucleic Acids Res. 1984 Dec 21;12(24):9287–9298. doi: 10.1093/nar/12.24.9287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nogi Y. GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae. J Bacteriol. 1986 Jan;165(1):101–106. doi: 10.1128/jb.165.1.101-106.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pan T., Coleman J. E. Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor. Proc Natl Acad Sci U S A. 1989 May;86(9):3145–3149. doi: 10.1073/pnas.86.9.3145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Parthun M. R., Jaehning J. A. Purification and characterization of the yeast transcriptional activator GAL4. J Biol Chem. 1990 Jan 5;265(1):209–213. [PubMed] [Google Scholar]
  48. Riley M. I., Hopper J. E., Johnston S. A., Dickson R. C. GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon. Mol Cell Biol. 1987 Feb;7(2):780–786. doi: 10.1128/mcb.7.2.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ruden D. M., Ma J., Ptashne M. No strict alignment is required between a transcriptional activator binding site and the "TATA box" of a yeast gene. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4262–4266. doi: 10.1073/pnas.85.12.4262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Salmeron J. M., Jr, Johnston S. A. Analysis of the Kluyveromyces lactis positive regulatory gene LAC9 reveals functional homology to, but sequence divergence from, the Saccharomyces cerevisiae GAL4 gene. Nucleic Acids Res. 1986 Oct 10;14(19):7767–7781. doi: 10.1093/nar/14.19.7767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schultz L. D., Hofmann K. J., Mylin L. M., Montgomery D. L., Ellis R. W., Hopper J. E. Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast. Gene. 1987;61(2):123–133. doi: 10.1016/0378-1119(87)90107-7. [DOI] [PubMed] [Google Scholar]
  52. Selleck S. B., Majors J. E. In vivo DNA-binding properties of a yeast transcription activator protein. Mol Cell Biol. 1987 Sep;7(9):3260–3267. doi: 10.1128/mcb.7.9.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Shimada H., Fukasawa T. Controlled transcription of the yeast regulatory gene GAL80. Gene. 1985;39(1):1–9. doi: 10.1016/0378-1119(85)90100-3. [DOI] [PubMed] [Google Scholar]
  54. Silver P. A., Keegan L. P., Ptashne M. Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5951–5955. doi: 10.1073/pnas.81.19.5951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sorger P. K., Pelham H. R. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell. 1988 Sep 9;54(6):855–864. doi: 10.1016/s0092-8674(88)91219-6. [DOI] [PubMed] [Google Scholar]
  56. Strick C. A., Fox T. D. Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader. Mol Cell Biol. 1987 Aug;7(8):2728–2734. doi: 10.1128/mcb.7.8.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Subbiah S., Harrison S. C. A method for multiple sequence alignment with gaps. J Mol Biol. 1989 Oct 20;209(4):539–548. doi: 10.1016/0022-2836(89)90592-5. [DOI] [PubMed] [Google Scholar]
  58. Webster N., Jin J. R., Green S., Hollis M., Chambon P. The yeast UASG is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator. Cell. 1988 Jan 29;52(2):169–178. doi: 10.1016/0092-8674(88)90505-3. [DOI] [PubMed] [Google Scholar]
  59. Wray L. V., Jr, Witte M. M., Dickson R. C., Riley M. I. Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):1111–1121. doi: 10.1128/mcb.7.3.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

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