Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Feb 1996; 16(2): 724–734.
PMCID: PMC231052

Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions.


Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity.

Full Text

The Full Text of this article is available as a PDF (620K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ansari AZ, Chael ML, O'Halloran TV. Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature. 1992 Jan 2;355(6355):87–89. [PubMed]
  • Ashar HR, Fejzo MS, Tkachenko A, Zhou X, Fletcher JA, Weremowicz S, Morton CC, Chada K. Disruption of the architectural factor HMGI-C: DNA-binding AT hook motifs fused in lipomas to distinct transcriptional regulatory domains. Cell. 1995 Jul 14;82(1):57–65. [PubMed]
  • Baldwin CT, Hoth CF, Amos JA, da-Silva EO, Milunsky A. An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome. Nature. 1992 Feb 13;355(6361):637–638. [PubMed]
  • Balling R, Deutsch U, Gruss P. undulated, a mutation affecting the development of the mouse skeleton, has a point mutation in the paired box of Pax 1. Cell. 1988 Nov 4;55(3):531–535. [PubMed]
  • Bernardi A, Gaillard C, Bernardi G. The specificity of five DNAases as studied by the analysis of 5'-terminal doublets. Eur J Biochem. 1975 Apr 1;52(3):451–457. [PubMed]
  • Bopp D, Burri M, Baumgartner S, Frigerio G, Noll M. Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila. Cell. 1986 Dec 26;47(6):1033–1040. [PubMed]
  • Buchman C, Skroch P, Dixon W, Tullius TD, Karin M. A single amino acid change in CUP2 alters its mode of DNA binding. Mol Cell Biol. 1990 Sep;10(9):4778–4787. [PMC free article] [PubMed]
  • Buchman C, Skroch P, Welch J, Fogel S, Karin M. The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein. Mol Cell Biol. 1989 Sep;9(9):4091–4095. [PMC free article] [PubMed]
  • Carri MT, Galiazzo F, Ciriolo MR, Rotilio G. Evidence for co-regulation of Cu,Zn superoxide dismutase and metallothionein gene expression in yeast through transcriptional control by copper via the ACE 1 factor. FEBS Lett. 1991 Jan 28;278(2):263–266. [PubMed]
  • Chalepakis G, Wijnholds J, Gruss P. Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains. Nucleic Acids Res. 1994 Aug 11;22(15):3131–3137. [PMC free article] [PubMed]
  • Culotta VC, Howard WR, Liu XF. CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J Biol Chem. 1994 Oct 14;269(41):25295–25302. [PubMed]
  • Deutsch U, Dressler GR, Gruss P. Pax 1, a member of a paired box homologous murine gene family, is expressed in segmented structures during development. Cell. 1988 May 20;53(4):617–625. [PubMed]
  • Dobi A, Dameron CT, Hu S, Hamer D, Winge DR. Distinct regions of Cu(I).ACE1 contact two spatially resolved DNA major groove sites. J Biol Chem. 1995 Apr 28;270(17):10171–10178. [PubMed]
  • Evans CF, Engelke DR, Thiele DJ. ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences. Mol Cell Biol. 1990 Jan;10(1):426–429. [PMC free article] [PubMed]
  • Fried M, Crothers DM. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. [PMC free article] [PubMed]
  • Fürst P, Hamer D. Cooperative activation of a eukaryotic transcription factor: interaction between Cu(I) and yeast ACE1 protein. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5267–5271. [PMC free article] [PubMed]
  • Fürst P, Hu S, Hackett R, Hamer D. Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein. Cell. 1988 Nov 18;55(4):705–717. [PubMed]
  • Geierstanger BH, Volkman BF, Kremer W, Wemmer DE. Short peptide fragments derived from HMG-I/Y proteins bind specifically to the minor groove of DNA. Biochemistry. 1994 May 3;33(17):5347–5355. [PubMed]
  • Gralla EB, Thiele DJ, Silar P, Valentine JS. ACE1, a copper-dependent transcription factor, activates expression of the yeast copper, zinc superoxide dismutase gene. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8558–8562. [PMC free article] [PubMed]
  • Halliwell B. Free radicals and antioxidants: a personal view. Nutr Rev. 1994 Aug;52(8 Pt 1):253–265. [PubMed]
  • Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990;186:1–85. [PubMed]
  • Hayes JJ, Tullius TD. The missing nucleoside experiment: a new technique to study recognition of DNA by protein. Biochemistry. 1989 Nov 28;28(24):9521–9527. [PubMed]
  • Hope IA, Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986 Sep 12;46(6):885–894. [PubMed]
  • Hoth CF, Milunsky A, Lipsky N, Sheffer R, Clarren SK, Baldwin CT. Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I). Am J Hum Genet. 1993 Mar;52(3):455–462. [PMC free article] [PubMed]
  • Hu S, Fürst P, Hamer D. The DNA and Cu binding functions of ACE1 are interdigitated within a single domain. New Biol. 1990 Jun;2(6):544–555. [PubMed]
  • Huibregtse JM, Engelke DR, Thiele DJ. Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences. Proc Natl Acad Sci U S A. 1989 Jan;86(1):65–69. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Jungmann J, Reins HA, Lee J, Romeo A, Hassett R, Kosman D, Jentsch S. MAC1, a nuclear regulatory protein related to Cu-dependent transcription factors is involved in Cu/Fe utilization and stress resistance in yeast. EMBO J. 1993 Dec 15;12(13):5051–5056. [PMC free article] [PubMed]
  • Köhrer K, Domdey H. Preparation of high molecular weight RNA. Methods Enzymol. 1991;194:398–405. [PubMed]
  • Lund T, Dahl KH, Mørk E, Holtlund J, Laland SG. The human chromosomal protein HMG I contains two identical palindrome amino acid sequences. Biochem Biophys Res Commun. 1987 Jul 31;146(2):725–730. [PubMed]
  • Majumdar A, Adhya S. Effect of ethylation of operator-phosphates on Gal repressor binding. DNA contortion by repressor. J Mol Biol. 1989 Jul 20;208(2):217–223. [PubMed]
  • O'Halloran TV. Transition metals in control of gene expression. Science. 1993 Aug 6;261(5122):715–725. [PubMed]
  • Predki PF, Sarkar B. Effect of replacement of "zinc finger" zinc on estrogen receptor DNA interactions. J Biol Chem. 1992 Mar 25;267(9):5842–5846. [PubMed]
  • Saccomanno CF, Bordonaro M, Chen JS, Nordstrom JL. A faster ribonuclease protection assay. Biotechniques. 1992 Dec;13(6):846–850. [PubMed]
  • Sorger PK, Pelham HR. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell. 1988 Sep 9;54(6):855–864. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Szczypka MS, Thiele DJ. A cysteine-rich nuclear protein activates yeast metallothionein gene transcription. Mol Cell Biol. 1989 Feb;9(2):421–429. [PMC free article] [PubMed]
  • Thiele DJ. ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene. Mol Cell Biol. 1988 Jul;8(7):2745–2752. [PMC free article] [PubMed]
  • Thiele DJ. Metal-regulated transcription in eukaryotes. Nucleic Acids Res. 1992 Mar 25;20(6):1183–1191. [PMC free article] [PubMed]
  • Thorvaldsen JL, Sewell AK, Tanner AM, Peltier JM, Pickering IJ, George GN, Winge DR. Mixed Cu+ and Zn2+ coordination in the DNA-binding domain of the AMT1 transcription factor from Candida glabrata. Biochemistry. 1994 Aug 16;33(32):9566–9577. [PubMed]
  • Xu W, Rould MA, Jun S, Desplan C, Pabo CO. Crystal structure of a paired domain-DNA complex at 2.5 A resolution reveals structural basis for Pax developmental mutations. Cell. 1995 Feb 24;80(4):639–650. [PubMed]
  • Zhang SP, Zubay G, Goldman E. Low-usage codons in Escherichia coli, yeast, fruit fly and primates. Gene. 1991 Aug 30;105(1):61–72. [PubMed]
  • Zhou P, Szczypka MS, Sosinowski T, Thiele DJ. Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor. Mol Cell Biol. 1992 Sep;12(9):3766–3775. [PMC free article] [PubMed]
  • Zhou P, Szczypka MS, Young R, Thiele DJ. A system for gene cloning and manipulation in the yeast Candida glabrata. Gene. 1994 May 3;142(1):135–140. [PubMed]
  • Zhou PB, Thiele DJ. Isolation of a metal-activated transcription factor gene from Candida glabrata by complementation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6112–6116. [PMC free article] [PubMed]
  • Zhou P, Thiele DJ. Rapid transcriptional autoregulation of a yeast metalloregulatory transcription factor is essential for high-level copper detoxification. Genes Dev. 1993 Sep;7(9):1824–1835. [PubMed]
  • Zieg J, Simon M. Analysis of the nucleotide sequence of an invertible controlling element. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4196–4200. [PMC free article] [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem Compound links
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links
  • Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...