Abstract
A positive regulatory genePDC2 required for expression of the enzyme pyruvate decarboxylase (PDC) in the yeastSaccharomyces cerevisiae has been identified and cloned. Thepdc2 mutant lacks pyruvate decarboxylase activity and is unable to grow on a medium containing glucose.PDC2 disruptants are viable on ethanol. ThePDC2 gene product is essential for transcription ofPDC1 andPDC5, the structural genes of pyruvate decarboxylase. ThePDC2 gene codes for a low-abundance mRNA of approximately 2.8 kb. Transformation of a wild-type strain with multiple copies of the promoter ofPDC1 leads to decreased pyruvate decarboxylase activity, presumably owing to titration oftrans-acting factors. Normal activity is restored by multiple copies ofPDC2, implicating involvement ofPDC2 in transcription ofPDC1. The deducedPDC2 protein (Pdc2p) sequence contains 925 amino acids, and is rich in asparagine and serine. We fused the DNA sequence encoding the N-terminal domain of Gal4p to the sequence encoding the C-terminal of Pdc2p; the hybrid protein (Gal4-Pdc2p) was able to activate transcription of theGAL1-lucZ fusion gene. The active domain consists of an unusual structure with a strikingly high asparagine content. We propose that this asparagine-rich domain represents a novel structural motif for transcriptional activation.PDC2 maps on chromosome IV betweencdc34 andarol; PDC1 is on the left arm of chromosome XII, linked topprl.
Similar content being viewed by others
References
Altslml S. F., Gish W., Miller W., Myers E. W. and Lipman D. J. 1990 Basic local alignment search tool.J. Mol. Biol. 215: 403–410
Baker H. V. 1986 Glycolytic gene expression inSaccharomyces cerevisiae: Nucleotide sequence ofGCR1 null mutants, and evidence for expression.Mol. Cell. Biol. 6: 3774–3784
Baker H. V. 1991GCR1 ofSaccharomyces cerevisiae encodes a DNA binding protein whose binding is abolished by mutations in the CTTCC sequence motif.Proc. Natl. Acad. Sci. USA 88: 9443–9447
Berger S. L., Pina B., Silverman N., Marcus G. A., Agapite J., Reiger J. L., Triezenberg S. J. and Guarente L. 1992 Genetic isolation of ADA2: a potential transcriptional adaptor required for function of certain acidic activation domains.Cell 70: 251–265
Bitter G. A., Chang K. K. H. and Egan K. M. 1991 A multi-component upstream activation sequence of theSaccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter.Mol. Gen. Genet. 231: 22–32
Brindle P. K., Holland J. P., Willet C. E., Innis M. A. and Holland M. J. 1990 Multiple factors bind the upstream activation sites of the yeast enolase genesEN01 andENO2: ABFI protein, like repressor activator protein RAP1, bindscis-acting sequences which modulate repression or activation of transcription.Mol. Cell. Biol. 10: 4872–4885
Buchman A., Kimberly W. J., Rhine J. and Kornberg R. D. 1988a Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences and telomers inSaccharomyces cerevisiae.Mol. Cell. Biol. 8: 210–225
Buchman A. R., Lue N. F. and Kornberg R. D. 1988b Connections between transcriptional activators, silencers, and telomers as revealed by functional analysis of yeast DNA-binding protein.Mol. Cell. Biol. 8: 5086–5099
Butler G., Dawes I. W. and McConnell D. J. 1990 TUF factor binds to the upstream region of the pyruvale decarboxylase structural genePDC1 ofSaccharomyces cerevisiae.Mol. Gen. Genet. 223: 449–456
Butler G. and McConnell D. J. 1988 Identification of an upstream activation site in the pyruvale decarboxylase structural genePDC1 ofSaccharomyces cerevisiae.Curr. Genet. 14: 405–412
Chasman D. I., Lue N. F., Buchman A. R., LaPointe J. W., Lorch Y. and Kornberg R. D. 1990 A yeast protein that influences the chromatin structure of UASG and functions as a powerful auxiliary gene activator.Genes Dev. 4: 503–514
Chien C. T., Bartel P. L., Sternglanz R. and Fields S. 1991 The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest.Proc. Natl. Acad. Sci. USA 87: 9578–9582
Clifton D. and Fraenkel D. G. 1981 Thegcr (glycolysis regulation) mutation ofSaccharomyces cerevisiae.J. Biol. Chem. 256: 13074–13078
Clifton D., Weinstock S. B. and Fraenkel D. G. 1978 Glycolysis mutants inSaccharomyces cerevisiae.Genetics 88: 1–11
Cohen R., Holland J. P. and Holland A. E. P. M. J. 1987 Transcription of the constitutively expressed yeast enolase geneENO1 is mediated by positive and negativecis-acting regulatory sequences.Mol. Cell. Biol. 7: 2753–2761
Courey A. J. and Tjian R. 1988 Analysis of Splin vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif.Cell 55: 887–898
Hanahan D. 1983 Studies on transformation ofE. coli with plasmids.J. Mol. Biol. 166: 557–580
Henry S. A., Donahue T. F. and Culbertson M. R. 1975 Selection of spontaneous mutants by inositol starvation in yeast.Mol. Gen. Genet. 143: 5–11
Hinnen A., Hicks J. B. and Fink G. R. 1978 Transformation of yeast.Proc. Natl. Acad. Sci. USA 75: 1929–1933
Hohmann S. 1991 Characterization ofPDC6, a third structural gene for pyruvate decarboxylase inSaccharomyces cerevisiae.J. Bacteriol. 173: 7963–7969
Hohmann S. 1993 Characterization ofPDC2, a gene necessary for the. high level expression of pyruvate decarboxylase structural genes inSaccharomyces cerevisiae.Mol. Gen. Genet. 241: 657–666
Hohmann S. and Cederberg H. 1990 Autoregulation may control the expression of pyruvate decarboxylase structural genesPDC1 andPDC5.Eur. J. Biochem. 188: 615–621
Holland M. J., Yokoi T., Holland J. P., Myambo K. and Innis M. A. 1987 TheGCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraklehyde-3-phosphate dehydrogenase gene families inSaccharomyces cerevisiae.Mol. Cell. Biol. 7: 813–820
Hope I. A. and Struhl K. 1986 Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast.Cell 46: 885–894
Huet J., Cottrelle P., Cool M. L., Thiele D., Marck C., Buhler J., Sentenac A. and Fromageot P. 1985 A general upstream binding factor for genes of the yeast translational apparatus.EMBO J. 4: 3539–3547
Huie M. A., Scott E. W., Drazinic C. M., Lopez M. C., Hornstra I. K., Yang T. P. and Baker H. V. 1992 Characterization of the DNA-binding activity of GCR1: In vivo evidence for two GCRl-binding sites in the upstream activation sequence ofTP1 ofSaccharomyces cerevisiae.Mol. Cell. Biol. 12: 2690–2700
Ito H., Jukucla Y., Murata K. and Kimura A. 1983 Transformation of intact yeast cells treated with alkali cations.J. Bacteriol. 153: 163–168
Kellermann E. and Hollenberg C. P. 1988 The glucose and ethanol-dependent regulation ofPDC1 fromSaccharomyces cerevisiae are controlled by two distinct promoter regions.Curr. Genet. 14: 337–344
Kim J.-H. and Powers S. 1991 Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated Ras function.Mol. Cell. Biol. 11: 3894–3904
Legrain P., Chapon C. and Galisson F. 1993 Interactions between PRP9 and SPP91 splicing factors identify a protein complex required in prespliceosome assembly.Genes Dev. 7: 1390–1399
Ma J. and Ptashne M. 1987 Deletion analysis of GAL4 defines two transcriptional activating segments.Cell 48: 847–853
Maitra P. K. and Lobo Z. 1971a A kinetic study of glycolytic enzyme synthesis in yeast.J. Biol. Chem. 246: 475–488
Maitra P. K. and Lobo Z. 1971b Control of glycolytic enzyme synthesis in yeast by products of the hexokinase reaction.J. Biol. Chem. 246: 489–499
Maitra P. K. and Lobo Z. 1978 Reversal of glycolysis in yeast.Arch. Biochem. Biophys. 185: 535–543
Miller J. H. 1972Experiments in molecular genetics (Cold Spring Harbor: Cold Spring Harbor Laboratory Press)
Moore P. A., Sagliocco F. A., Wood R. M. and Brown A. J. P. 1991 Yeast glycolytic mRNA are differentially regulated.Mol. Cell. Biol. 11: 5330–5337
Nishizawa M., Araki R. and Teranishi Y. 1989 Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvale kinase gene of the yeastSaccharomyces cerevisiae.Mol. Cell. Biol. 9: 442–451
Sambrook J., Fritsch E. F. and Maniatis T. 1989Molecular cloning: A laboratory manual, second edition (Cold Spring Harbor: Cold Spring Harbor Laboratory Press)
Santangelo G. M. and Tornow J. 1990 Efficient transcription of the glycolytic geneADH1 and three translational component genes requiresGCR1 product, which can act through TUF/GRF/RAP binding sites.Mol. Cell. Biol. 10: 859–862
Schaaff I., Green J. B. A., Gozablo D. and Hohmann S. 1989 A deletion of thePDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations.Curr. Genet. 15: 75–91
Schmitl H. D., Ciriacy M. and Zimmermann F. K. 1983 The synthesis of yeast pyruvate decarboxylase is regulated by large variations in the messenger RNA level.Mol. Gen. Genet. 192: 247–252
Schmitt H. D. and Zimmermann F. K. 1982 Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis.J. Bacteriol. 151: 1146–1152
Scott E. W. and Baker H. V. 1993 Concerted action of the transcriptional activators REB1, RAP1, GCR1 in the high-level expression of the glycolytic geneTPII.Mol. Cell. Biol. 13: 534–550
Seeboth P. G., Bohnsack K. and Hollenberg C. P. 1990pdcl mutants ofSaccharomyces cerevisiae give evidence for an additional structuralPDC gene: Cloning ofPDC5, a gene homologous toPDC1.J. Bacteriol. 172: 678–685
Sharp P. M. and Cowe E. 1991 Synonymous codon usage inSaccharomyces cerevisiae.Yeast 7: 657–678
Sherman F., Fink G. R. and Hicks J. B. 1979Methods in yeast genetics (Cold Spring Harbor: Cold Spring Harbor Laboratory Press)
Sherman F., Fink G. R. and Hicks J. B. 1986Methods in yeast genetics (Cold Spring Harbor: Cold Spring Harbor Laboratory Press)
Shore D. and Nasmyth K. 1987 Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements.Cell 51: 721–732
Stanway C., Mellor J., Ogden J. E., Kingsman A. J. and Kingsman S. M. 1987 The UAS of the yeastPGK gene contains functionally distinct domains.Nucleic Acids Res. 15: 6855–6873
Strathmann M., Hamilton B. A., Mayeda C. A., Simon M. I., Meyerowitz E. M. and Palazollo M. J. 1991 Transposon-facilitated DNA sequencing.Proc. Natl. Acad. Sci. USA 88: 1247–1250
Uemura H. and Fraenkel D. G. 1990gcr2, a new mutation affecting glycolytic gene expression inSaccharomyces cerevisiae.Mol. Cell. Biol. 10: 6389–6396
Uemura H. and Jigami Y. 1992 Role of GCR2 in transcriptional activation of yeast glycolytic genes.Mol. Cell. Biol. 12: 3834–3842
Uemura H., Shiba T., Paterson M., Jigami Y. and Tanaka H. 1986 Identification of a sequence containing the positive regulatory region ofSaccharomyces cerevisiae geneENO1.Gene 45: 67–75
Vollrath D., Davis R. W., Connelly C. and Heiter P. 1988 Physical mapping of large DNA by chromosome fragmentation.Proc. Natl. Acad. Sci. USA 85: 6027–6031
Walton E. F., Carter B. L. A. and Pringle J. R. 1979 An enrichment method for temperature-sensitive and auxotrophic mutants of yeast.Mol. Gen. Genet. 171: 111–114
Wright A. P. H., Png H.-L. and Hartley B. S. 1989 Identification of a new gene required for full pyruvate decarboxylase activity inSaccharomyces cerevisiae.Curr. Genet. 15: 171–175
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Raghuram, V., Lobo, Z. & Maitra, P.K. PDC2, a yeast gene essential for synthesis of pyruvate decarboxylase, encodes a novel transcription factor. J. Genet. 73, 17–32 (1994). https://doi.org/10.1007/BF02927930
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02927930