Summary
The gene AR07 encodes the monofunctional enzyme chorismate mutase, a branch point enzyme in the aromatic amino acid biosynthetic pathway in Saccharomyces cerevisiae. We investigated the transcription of the AR07 gene. Three 5′ ends at positions − 36, − 56 and − 73 and the 3′ end of the transcripts 146 bp downstream of the translational stop codon were mapped. As in the promoters of other aromatic amino acid biosynthetic genes, a recognition element for the GCN4 transcriptional activator of amino acid biosynthesis is located 425 base pairs (bp) upstream of the first transcriptional start point. This element binds GCN4 specifically in vitro. Northern analysis and determination of the specific enzyme activity reveals however, that the element is not sufficient to mediate transcriptional regulation by GCN4 in vivo. We thus suggest that in addition to a consensus sequence capable of binding the GCN4 protein other factors like, for example, chromatin structure, determine whether a recognition site for a transcription factor functions as an upstream activation sequence.
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Aebi M, Furter R, Prantl F, Niederberger P, Hütter R (1984) Structure and function of the TRP3 gene of Saccharomyces cerevisiae: Analysis of transcription, promoter sequence, and sequence coding for a glutamine amidotransferase. Curr Genet 8:165–172
Arndt KT, Fink GR (1986) GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5′ TGACTC 3′ sequences. Proc Natl Acad Sci USA 83:8516–8520
Arndt KT, Styles CA, Fink GR (1989) A suppressor of a HIS4 transcriptional defect encodes a protein with homology to the catalytic subunit of protein phosphatases. Cell 56:527–537
Aviv H, Leder P (1972) Purification of biologically active globin mRNA by chromatography on oligothymidilic cellulose. Proc Natl Acad Sci USA 69:1408–1412
Braus G, Luger K, Paravicini G, Schmidheini T, Kirschner K, Hütter R (1988a) The role of the TRP1 gene in yeast tryptophan biosynthesis. J Biol Chem 263:7868–7875
Braus G, Paravicini G, Hütter R (1988b) A consensus transcription termination sequence in the promoter region is necessary for efficient gene expression of the TRP1 gene of Saccharomyces cerevisiae. Mol Gen Genet 212:495–504
Braus G, Mösch HU, Vogel K, Hinnen A, Hütter R (1989) Interpathway regulation fo the TRP4 gene of yeast. EMBO J 8:939–945
Donahue TF, Farabaugh PJ, Fink GR (1982) The nucleotide sequence of the HIS4 region of yeast. Gene 18:47–59
Donahue TF, Daves RS, Lucchini G, Fink GR (1983) A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast. Cell 32:89–98
Favoloro J, Treisman R, Kamen R (1980) Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol 65:718–749
Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266–267
Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9:6505–6525
Furter R, Paravicini G, Aebi M, Braus G, Prantl F, Niederberger P, Hütter R (1986) The TRP4 gene of Saccharomyces cerevisiae. Isolation and structural analysis. Nucleic Acids Res 14:6357–6373
Furter R, Braus G, Paravicini G, Mösch HU, Niederberger P, Hütter R (1988) Regulation of the TRP4 gene of Saccharomyces cerevisiae at the transcriptional level and functional analysis of its promoter. Mol Gen Genet 211:168–175
Galas D, Schmitz A (1978) DNase I footprinting: A simple method for the detection of protein-binding specificity. Nucleic Acids Res 5:3157–3170
Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: Application to components fo the Escherichia coli lactose operon regulatory system. Nucleic Acids Res 9:3047–3060
Guarente L, Hoar E (1984) Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the “TATA” box. Proc Natl Acad Sci USA 81:7860–7864
Harshman KD, Moye-Rowley WS, Parker CS (1988) Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4. Cell 53:321–330
Hill DE, Hope IA, Macke JP, Struhl K (1986) Saturation mutagenesis of the yeast HIS3 regulatory site: Requirements for transcriptional induction and for binding by GCN4 activator protein. Science 234:451–457
Hinnebusch AG (1988) Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev 52:248–273
Hinnebusch AG, Fink GR (1983) Positive regulation in the general amino acid control of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 80:5374–5378
Hinnebusch AG, Lucchini G, Fink GR (1985) A synthetic HIS4 regulatory element confers general amino acid control on the cytochrome c gene (CYC1) of yeast. Proc Natl Acad Sci USA 82:498–502
Hope IA, Struhl K (1985) GCN4 protein, synthesized in vitro, binds to HIS3 regulatory sequences: Implications for the general control of amino acid biosynthetic genes in yeast. Cell 43:177–188
Hope IA, Struhl K (1987) GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J 6:2781–2784
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Kassavetis GA, Geiduschek EP (1982) Bacteriophage T4 late promoters: mapping 5′-ends of T4 gene 23 S mRNAs. EMBO J 1:107–114
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Miozzari G, Niederberger P, Hütter R (1978) Tryptophan biosynthesis in Saccharomyces cerevisiae: Control of the flux through the pathway. J Bacteriol 134:48–59
Mösch HU, Graf R, Schmidheini T, Braus G (1990) Three GCN4 responsive elements act synergistically as upstream and as TATA-like elements in the yeast TRP4 promoter. EMBO J 9:2951–2957
Ng R, Abelson J (1980) Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 77:3912–3916
Paravicini G, Mösch HU, Schmidheini T, Braus G (1989) The general control activator protein GCN4 is essential for a basal level of ARO3 gene expression in Saccharomyces cerevisiae. Mol Cell Biol 9:144–151
Schmidheini T, Sperisen P, Paravicini G, Hütter R, Braus G (1989) A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase in Saccharomyces cerevisiae. J Bacteriol 171:1245–1253
Schmidheini T, Mösch HU, Evans JNS, Braus G (1990) The yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution. Biochemistry 29:3660–3668
Schürch AR, Miozzari G, Hütter R (1974) Regulation of tryptophan biosynthesis in Saccharomyces cerevisiae: mode of action of 5-methyltryptophan and 5-methyltryptophan-sensitive mutants. J Bacteriol 117:1131–1140
Struhl K (1982) The yeast HIS3 promoter contains at least two distinct elements. Proc Natl Acad Sci USA 79:7385–7389
Struhl K (1987a) Promoters, activator proteins, and the mechanism of transcriptional initiation in yeast. Cell 49:295–297
Struhl K (1987b) The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous. Cell 50:841–846
Zalkin H, Yanofsky C (1982) Yeast gene TRP5: Structure, function, regulation. J Biol Chem 257:1491–1500
Zalkin h, Paluh JL, van Cleemput M, Moye WS, Yanofsky C (1984) Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase:indole-3-glycerol phosphate synthase. J Biol Chem 259:3985–3992
Zaret KS, Sherman F (1982) DNA sequences required for efficient transcription termination in yeast. Cell 28:563–573
Zitomer RS, Hall BD (1976) Yeast cytochrome c messenger RNA. J Biol Chem 251:6320–6326
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Communicated by H. Hennecke
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Schmidheini, T., Mösch, HU., Graf, R. et al. A GCN4 protein recognition element is not sufficient for GCN4-dependent regulation of transcription in the ARO7 promoter of Saccharomyces cerevisiae . Molec. Gen. Genet. 224, 57–64 (1990). https://doi.org/10.1007/BF00259451
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DOI: https://doi.org/10.1007/BF00259451