Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1997 Oct;147(2):521–532. doi: 10.1093/genetics/147.2.521

The Role of Gcr1p in the Transcriptional Activation of Glycolytic Genes in Yeast Saccharomyces Cerevisiae

H Uemura 1, M Koshio 1, Y Inoue 1, M C Lopez 1, H V Baker 1
PMCID: PMC1208175  PMID: 9335590

Abstract

To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites). The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene. As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity. Likewise, a lone CT-box did not show any UAS activity. By contrast, oligonucleotides containing tandem CT-boxes but no RPG-box conferred strong promoter activity. This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5' noncoding region. However, it was dependent on both GCR1 and GCR2. These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p. Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box. Five mutants had single mutations in Gcr1p's DNA-binding domain and displayed slightly higher affinity for the CT-box. These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker H. V. GCR1 of Saccharomyces cerevisiae encodes a DNA binding protein whose binding is abolished by mutations in the CTTCC sequence motif. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9443–9447. doi: 10.1073/pnas.88.21.9443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bitter G. A., Chang K. K., Egan K. M. A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter. Mol Gen Genet. 1991 Dec;231(1):22–32. doi: 10.1007/BF00293817. [DOI] [PubMed] [Google Scholar]
  3. Brindle P. K., Holland J. P., Willett C. E., Innis M. A., Holland M. J. Multiple factors bind the upstream activation sites of the yeast enolase genes ENO1 and ENO2: ABFI protein, like repressor activator protein RAP1, binds cis-acting sequences which modulate repression or activation of transcription. Mol Cell Biol. 1990 Sep;10(9):4872–4885. doi: 10.1128/mcb.10.9.4872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Butler G., Dawes I. W., McConnell D. J. TUF factor binds to the upstream region of the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae. Mol Gen Genet. 1990 Sep;223(3):449–456. doi: 10.1007/BF00264453. [DOI] [PubMed] [Google Scholar]
  6. Carmen A. A., Holland M. J. The upstream repression sequence from the yeast enolase gene ENO1 is a complex regulatory element that binds multiple trans-acting factors including REB1. J Biol Chem. 1994 Apr 1;269(13):9790–9797. [PubMed] [Google Scholar]
  7. Chambers A., Stanway C., Tsang J. S., Henry Y., Kingsman A. J., Kingsman S. M. ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeast glycolytic genes PGK and PYK1. Nucleic Acids Res. 1990 Sep 25;18(18):5393–5399. doi: 10.1093/nar/18.18.5393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chambers A., Tsang J. S., Stanway C., Kingsman A. J., Kingsman S. M. Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1. Mol Cell Biol. 1989 Dec;9(12):5516–5524. doi: 10.1128/mcb.9.12.5516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Clifton D., Weinstock S. B., Fraenkel D. G. Glycolysis mutants in Saccharomyces cerevisiae. Genetics. 1978 Jan;88(1):1–11. doi: 10.1093/genetics/88.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deminoff S. J., Tornow J., Santangelo G. M. Unigenic evolution: a novel genetic method localizes a putative leucine zipper that mediates dimerization of the Saccharomyces cerevisiae regulator Gcr1p. Genetics. 1995 Dec;141(4):1263–1274. doi: 10.1093/genetics/141.4.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Devlin C., Tice-Baldwin K., Shore D., Arndt K. T. RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene. Mol Cell Biol. 1991 Jul;11(7):3642–3651. doi: 10.1128/mcb.11.7.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drazinic C. M., Smerage J. B., López M. C., Baker H. V. Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p). Mol Cell Biol. 1996 Jun;16(6):3187–3196. doi: 10.1128/mcb.16.6.3187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gill G., Ptashne M. Mutants of GAL4 protein altered in an activation function. Cell. 1987 Oct 9;51(1):121–126. doi: 10.1016/0092-8674(87)90016-x. [DOI] [PubMed] [Google Scholar]
  15. Gilson E., Roberge M., Giraldo R., Rhodes D., Gasser S. M. Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites. J Mol Biol. 1993 May 20;231(2):293–310. doi: 10.1006/jmbi.1993.1283. [DOI] [PubMed] [Google Scholar]
  16. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  17. Henry Y. A., López M. C., Gibbs J. M., Chambers A., Kingsman S. M., Baker H. V., Stanway C. A. The yeast protein Gcr1p binds to the PGK UAS and contributes to the activation of transcription of the PGK gene. Mol Gen Genet. 1994 Nov 15;245(4):506–511. doi: 10.1007/BF00302263. [DOI] [PubMed] [Google Scholar]
  18. Hess B., Boiteux A., Krüger J. Cooperation of glycolytic enzymes. Adv Enzyme Regul. 1969;7:149–167. doi: 10.1016/0065-2571(69)90016-8. [DOI] [PubMed] [Google Scholar]
  19. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  20. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  21. Huet J., Sentenac A. TUF, the yeast DNA-binding factor specific for UASrpg upstream activating sequences: identification of the protein and its DNA-binding domain. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3648–3652. doi: 10.1073/pnas.84.11.3648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huie M. A., Baker H. V. DNA-binding properties of the yeast transcriptional activator, Gcr1p. Yeast. 1996 Mar 30;12(4):307–317. doi: 10.1002/(sici)1097-0061(19960330)12:4<307::aid-yea912>3.0.co;2-c. [DOI] [PubMed] [Google Scholar]
  23. Huie M. A., Scott E. W., Drazinic C. M., Lopez M. C., Hornstra I. K., Yang T. P., Baker H. V. Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae. Mol Cell Biol. 1992 Jun;12(6):2690–2700. doi: 10.1128/mcb.12.6.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lustig A. J., Kurtz S., Shore D. Involvement of the silencer and UAS binding protein RAP1 in regulation of telomere length. Science. 1990 Oct 26;250(4980):549–553. doi: 10.1126/science.2237406. [DOI] [PubMed] [Google Scholar]
  26. Morrow B. E., Johnson S. P., Warner J. R. Proteins that bind to the yeast rDNA enhancer. J Biol Chem. 1989 May 25;264(15):9061–9068. [PubMed] [Google Scholar]
  27. Nishizawa M., Araki R., Teranishi Y. Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):442–451. doi: 10.1128/mcb.9.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  29. Packham E. A., Graham I. R., Chambers A. The multifunctional transcription factors Abf1p, Rap1p and Reb1p are required for full transcriptional activation of the chromosomal PGK gene in Saccharomyces cerevisiae. Mol Gen Genet. 1996 Feb 25;250(3):348–356. doi: 10.1007/BF02174393. [DOI] [PubMed] [Google Scholar]
  30. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  31. Scott E. W., Allison H. E., Baker H. V. Characterization of TPI gene expression in isogeneic wild-type and gcr1-deletion mutant strains of Saccharomyces cerevisiae. Nucleic Acids Res. 1990 Dec 11;18(23):7099–7107. doi: 10.1093/nar/18.23.7099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scott E. W., Baker H. V. Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI. Mol Cell Biol. 1993 Jan;13(1):543–550. doi: 10.1128/mcb.13.1.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shore D., Nasmyth K. Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell. 1987 Dec 4;51(5):721–732. doi: 10.1016/0092-8674(87)90095-x. [DOI] [PubMed] [Google Scholar]
  34. Shore D. RAP1: a protean regulator in yeast. Trends Genet. 1994 Nov;10(11):408–412. doi: 10.1016/0168-9525(94)90058-2. [DOI] [PubMed] [Google Scholar]
  35. Stanway C. A., Chambers A., Kingsman A. J., Kingsman S. M. Characterization of the transcriptional potency of sub-elements of the UAS of the yeast PGK gene in a PGK mini-promoter. Nucleic Acids Res. 1989 Nov 25;17(22):9205–9218. doi: 10.1093/nar/17.22.9205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Suzuki M., Brenner S. E. Classification of multi-helical DNA-binding domains and application to predict the DBD structures of sigma factor, LysR, OmpR/PhoB, CENP-B, Rapl, and Xy1S/Ada/AraC. FEBS Lett. 1995 Sep 25;372(2-3):215–221. doi: 10.1016/0014-5793(95)00988-l. [DOI] [PubMed] [Google Scholar]
  37. Tornow J., Santangelo G. The GCR1 gene of Saccharomyces cerevisiae is a split gene with an unusually long intron. Genetics. 1994 Nov;138(3):973–974. doi: 10.1093/genetics/138.3.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Uemura H., Fraenkel D. G. gcr2, a new mutation affecting glycolytic gene expression in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6389–6396. doi: 10.1128/mcb.10.12.6389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Uemura H., Jigami Y. GCR3 encodes an acidic protein that is required for expression of glycolytic genes in Saccharomyces cerevisiae. J Bacteriol. 1992 Sep;174(17):5526–5532. doi: 10.1128/jb.174.17.5526-5532.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Uemura H., Jigami Y. Mutations in GCR1, a transcriptional activator of Saccharomyces cerevisiae glycolytic genes, function as suppressors of gcr2 mutations. Genetics. 1995 Feb;139(2):511–521. doi: 10.1093/genetics/139.2.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Uemura H., Jigami Y. Role of GCR2 in transcriptional activation of yeast glycolytic genes. Mol Cell Biol. 1992 Sep;12(9):3834–3842. doi: 10.1128/mcb.12.9.3834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Uemura H., Shiba T., Paterson M., Jigami Y., Tanaka H. Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene ENO1. Gene. 1986;45(1):67–75. doi: 10.1016/0378-1119(86)90133-2. [DOI] [PubMed] [Google Scholar]
  43. Uemura H., Wickner R. B. Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein. Mol Cell Biol. 1988 Feb;8(2):938–944. doi: 10.1128/mcb.8.2.938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Willett C. E., Gelfman C. M., Holland M. J. A complex regulatory element from the yeast gene ENO2 modulates GCR1-dependent transcriptional activation. Mol Cell Biol. 1993 Apr;13(4):2623–2633. doi: 10.1128/mcb.13.4.2623. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES