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. 1995 Aug;15(8):4021–4030. doi: 10.1128/mcb.15.8.4021

Detection of leucine-independent DNA site occupancy of the yeast Leu3p transcriptional activator in vivo.

C R Kirkpatrick 1, P Schimmel 1
PMCID: PMC230641  PMID: 7623798

Abstract

The product of the Saccharomyces cerevisiae LEU3 gene, Leu3p, is a transcriptional activator which regulates leucine biosynthesis in response to intracellular levels of leucine through the biosynthetic intermediate alpha-isopropylmalate. We devised a novel assay to examine the DNA site occupancy of Leu3p under different growth conditions, using a reporter gene with internal Leu3p-binding sites. Expression of the reporter is inhibited by binding of nuclear Leu3p to these sites; inhibition is dependent on the presence of the sites in the reporter, on the integrity of the Leu3p DNA-binding domain, and, surprisingly, on the presence of a transcriptional activation domain in the inhibiting protein. By this assay, Leu3p was found to occupy its binding site under all conditions tested, including high and low levels of leucine and in the presence and absence of alpha-isopropylmalate. The localization of Leu3p to the nucleus was confirmed by immunofluorescence staining of cells expressing epitope-tagged Leu3p derivatives. We conclude that Leu3p regulates transcription in vivo without changing its intracellular localization and DNA site occupancy.

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Selected References

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  1. Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axelrod J. D., Majors J., Brandriss M. C. Proline-independent binding of PUT3 transcriptional activator protein detected by footprinting in vivo. Mol Cell Biol. 1991 Jan;11(1):564–567. doi: 10.1128/mcb.11.1.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baeuerle P. A., Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. doi: 10.1016/0092-8674(88)90382-0. [DOI] [PubMed] [Google Scholar]
  4. Bai Y. L., Kohlhaw G. B. Manipulation of the 'zinc cluster' region of transcriptional activator LEU3 by site-directed mutagenesis. Nucleic Acids Res. 1991 Nov 11;19(21):5991–5997. doi: 10.1093/nar/19.21.5991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Berger S. L., Piña B., Silverman N., Marcus G. A., Agapite J., Regier J. L., Triezenberg S. J., Guarente L. Genetic isolation of ADA2: a potential transcriptional adaptor required for function of certain acidic activation domains. Cell. 1992 Jul 24;70(2):251–265. doi: 10.1016/0092-8674(92)90100-q. [DOI] [PubMed] [Google Scholar]
  7. Brent R., Ptashne M. A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene. Nature. 1984 Dec 13;312(5995):612–615. doi: 10.1038/312612a0. [DOI] [PubMed] [Google Scholar]
  8. Brisco P. R., Cunningham T. S., Kohlhaw G. B. Cloning, disruption and chromosomal mapping of yeast LEU3, a putative regulatory gene. Genetics. 1987 Jan;115(1):91–99. doi: 10.1093/genetics/115.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brisco P. R., Kohlhaw G. B. Regulation of yeast LEU2. Total deletion of regulatory gene LEU3 unmasks GCN4-dependent basal level expression of LEU2. J Biol Chem. 1990 Jul 15;265(20):11667–11675. [PubMed] [Google Scholar]
  10. Carlson M., Laurent B. C. The SNF/SWI family of global transcriptional activators. Curr Opin Cell Biol. 1994 Jun;6(3):396–402. doi: 10.1016/0955-0674(94)90032-9. [DOI] [PubMed] [Google Scholar]
  11. Cress W. D., Triezenberg S. J. Critical structural elements of the VP16 transcriptional activation domain. Science. 1991 Jan 4;251(4989):87–90. doi: 10.1126/science.1846049. [DOI] [PubMed] [Google Scholar]
  12. Deuschle U., Hipskind R. A., Bujard H. RNA polymerase II transcription blocked by Escherichia coli lac repressor. Science. 1990 Apr 27;248(4954):480–483. doi: 10.1126/science.2158670. [DOI] [PubMed] [Google Scholar]
  13. Dingwall C., Laskey R. A. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. doi: 10.1146/annurev.cb.02.110186.002055. [DOI] [PubMed] [Google Scholar]
  14. Dohrmann P. R., Butler G., Tamai K., Dorland S., Greene J. R., Thiele D. J., Stillman D. J. Parallel pathways of gene regulation: homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase. Genes Dev. 1992 Jan;6(1):93–104. doi: 10.1101/gad.6.1.93. [DOI] [PubMed] [Google Scholar]
  15. Drain P., Schimmel P. Multiple new genes that determine activity for the first step of leucine biosynthesis in Saccharomyces cerevisiae. Genetics. 1988 May;119(1):13–20. doi: 10.1093/genetics/119.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Friden P., Schimmel P. LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence. Mol Cell Biol. 1988 Jul;8(7):2690–2697. doi: 10.1128/mcb.8.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Gill G., Ptashne M. Negative effect of the transcriptional activator GAL4. Nature. 1988 Aug 25;334(6184):721–724. doi: 10.1038/334721a0. [DOI] [PubMed] [Google Scholar]
  20. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  21. Hinnebusch A. G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev. 1988 Jun;52(2):248–273. doi: 10.1128/mr.52.2.248-273.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jakobsen B. K., Pelham H. R. Constitutive binding of yeast heat shock factor to DNA in vivo. Mol Cell Biol. 1988 Nov;8(11):5040–5042. doi: 10.1128/mcb.8.11.5040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keegan L., Gill G., Ptashne M. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science. 1986 Feb 14;231(4739):699–704. doi: 10.1126/science.3080805. [DOI] [PubMed] [Google Scholar]
  24. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  25. Kraulis P. J., Raine A. R., Gadhavi P. L., Laue E. D. Structure of the DNA-binding domain of zinc GAL4. Nature. 1992 Apr 2;356(6368):448–450. doi: 10.1038/356448a0. [DOI] [PubMed] [Google Scholar]
  26. Leuther K. K., Salmeron J. M., Johnston S. A. Genetic evidence that an activation domain of GAL4 does not require acidity and may form a beta sheet. Cell. 1993 Feb 26;72(4):575–585. doi: 10.1016/0092-8674(93)90076-3. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Nasmyth K., Adolf G., Lydall D., Seddon A. The identification of a second cell cycle control on the HO promoter in yeast: cell cycle regulation of SW15 nuclear entry. Cell. 1990 Aug 24;62(4):631–647. doi: 10.1016/0092-8674(90)90110-z. [DOI] [PubMed] [Google Scholar]
  30. Pavco P. A., Steege D. A. Elongation by Escherichia coli RNA polymerase is blocked in vitro by a site-specific DNA binding protein. J Biol Chem. 1990 Jun 15;265(17):9960–9969. [PubMed] [Google Scholar]
  31. Pfeifer K., Arcangioli B., Guarente L. Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene. Cell. 1987 Apr 10;49(1):9–18. doi: 10.1016/0092-8674(87)90750-1. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Picard D., Yamamoto K. R. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. doi: 10.1002/j.1460-2075.1987.tb02654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Remboutsika E., Kohlhaw G. B. Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach. Mol Cell Biol. 1994 Aug;14(8):5547–5557. doi: 10.1128/mcb.14.8.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rotenberg M. O., Woolford J. L., Jr Tripartite upstream promoter element essential for expression of Saccharomyces cerevisiae ribosomal protein genes. Mol Cell Biol. 1986 Feb;6(2):674–687. doi: 10.1128/mcb.6.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Silve S., Volland C., Garnier C., Jund R., Chevallier M. R., Haguenauer-Tsapis R. Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein. Mol Cell Biol. 1991 Feb;11(2):1114–1124. doi: 10.1128/mcb.11.2.1114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Sze J. Y., Remboutsika E., Kohlhaw G. B. Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions. Mol Cell Biol. 1993 Sep;13(9):5702–5709. doi: 10.1128/mcb.13.9.5702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sze J. Y., Woontner M., Jaehning J. A., Kohlhaw G. B. In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate. Science. 1992 Nov 13;258(5085):1143–1145. doi: 10.1126/science.1439822. [DOI] [PubMed] [Google Scholar]
  42. Taylor W. E., Young E. T. cAMP-dependent phosphorylation and inactivation of yeast transcription factor ADR1 does not affect DNA binding. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4098–4102. doi: 10.1073/pnas.87.11.4098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Triezenberg S. J., Kingsbury R. C., McKnight S. L. Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 1988 Jun;2(6):718–729. doi: 10.1101/gad.2.6.718. [DOI] [PubMed] [Google Scholar]
  44. Van Hoy M., Leuther K. K., Kodadek T., Johnston S. A. The acidic activation domains of the GCN4 and GAL4 proteins are not alpha helical but form beta sheets. Cell. 1993 Feb 26;72(4):587–594. doi: 10.1016/0092-8674(93)90077-4. [DOI] [PubMed] [Google Scholar]
  45. Zhou K. M., Bai Y. L., Kohlhaw G. B. Yeast regulatory protein LEU3: a structure-function analysis. Nucleic Acids Res. 1990 Jan 25;18(2):291–298. doi: 10.1093/nar/18.2.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhou K. M., Kohlhaw G. B. Transcriptional activator LEU3 of yeast. Mapping of the transcriptional activation function and significance of activation domain tryptophans. J Biol Chem. 1990 Oct 15;265(29):17409–17412. [PubMed] [Google Scholar]
  47. Zhou K., Brisco P. R., Hinkkanen A. E., Kohlhaw G. B. Structure of yeast regulatory gene LEU3 and evidence that LEU3 itself is under general amino acid control. Nucleic Acids Res. 1987 Jul 10;15(13):5261–5273. doi: 10.1093/nar/15.13.5261. [DOI] [PMC free article] [PubMed] [Google Scholar]

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