Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 May;12(5):2302–2314. doi: 10.1128/mcb.12.5.2302

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

J D Trawick 1, N Kraut 1, F R Simon 1, R O Poyton 1
PMCID: PMC364402  PMID: 1314953

Abstract

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

Full text

PDF
2302

Images in this article

2308Image
on p.2308
2309Image
on p.2309
2309Image
on p.2309
2310Image
on p.2310
2310Image
on p.2310
2310Image
on p.2310
2311Image
on p.2311
2311Image
on p.2311
2311Image
on p.2311

Selected References

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

  1. Arcangioli B., Lescure B. Identification of proteins involved in the regulation of yeast iso- 1-cytochrome C expression by oxygen. EMBO J. 1985 Oct;4(10):2627–2633. doi: 10.1002/j.1460-2075.1985.tb03980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buchman A. R., Kimmerly W. J., Rine J., Kornberg R. D. Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jan;8(1):210–225. doi: 10.1128/mcb.8.1.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buchman A. R., Kornberg R. D. A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors. Mol Cell Biol. 1990 Mar;10(3):887–897. doi: 10.1128/mcb.10.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlson M., Osmond B. C., Neigeborn L., Botstein D. A suppressor of SNF1 mutations causes constitutive high-level invertase synthesis in yeast. Genetics. 1984 May;107(1):19–32. doi: 10.1093/genetics/107.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Celenza J. L., Carlson M. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science. 1986 Sep 12;233(4769):1175–1180. doi: 10.1126/science.3526554. [DOI] [PubMed] [Google Scholar]
  7. Celenza J. L., Carlson M. Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein. Mol Cell Biol. 1989 Nov;9(11):5034–5044. doi: 10.1128/mcb.9.11.5034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cumsky M. G., Ko C., Trueblood C. E., Poyton R. O. Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2235–2239. doi: 10.1073/pnas.82.8.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dorsman J. C., van Heeswijk W. C., Grivell L. A. Identification of two factors which bind to the upstream sequences of a number of nuclear genes coding for mitochondrial proteins and to genetic elements important for cell division in yeast. Nucleic Acids Res. 1988 Aug 11;16(15):7287–7301. doi: 10.1093/nar/16.15.7287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dorsman J. C., van Heeswijk W. C., Grivell L. A. Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation. Nucleic Acids Res. 1990 May 11;18(9):2769–2776. doi: 10.1093/nar/18.9.2769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erecińska M., Wilson D. F. Regulation of cellular energy metabolism. J Membr Biol. 1982;70(1):1–14. doi: 10.1007/BF01871584. [DOI] [PubMed] [Google Scholar]
  12. Forsburg S. L., Guarente L. Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev. 1989 Aug;3(8):1166–1178. doi: 10.1101/gad.3.8.1166. [DOI] [PubMed] [Google Scholar]
  13. Forsburg S. L., Guarente L. Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site. Mol Cell Biol. 1988 Feb;8(2):647–654. doi: 10.1128/mcb.8.2.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gollub E. G., Liu K. P., Dayan J., Adlersberg M., Sprinson D. B. Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene. J Biol Chem. 1977 May 10;252(9):2846–2854. [PubMed] [Google Scholar]
  16. Guarente L., Lalonde B., Gifford P., Alani E. Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae. Cell. 1984 Feb;36(2):503–511. doi: 10.1016/0092-8674(84)90243-5. [DOI] [PubMed] [Google Scholar]
  17. Guarente L., Mason T. Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell. 1983 Apr;32(4):1279–1286. doi: 10.1016/0092-8674(83)90309-4. [DOI] [PubMed] [Google Scholar]
  18. Guarente L., Ptashne M. Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2199–2203. doi: 10.1073/pnas.78.4.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hahn S., Pinkham J., Wei R., Miller R., Guarente L. The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts. Mol Cell Biol. 1988 Feb;8(2):655–663. doi: 10.1128/mcb.8.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Halfter H., Müller U., Winnacker E. L., Gallwitz D. Isolation and DNA-binding characteristics of a protein involved in transcription activation of two divergently transcribed, essential yeast genes. EMBO J. 1989 Oct;8(10):3029–3037. doi: 10.1002/j.1460-2075.1989.tb08453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hamil K. G., Nam H. G., Fried H. M. Constitutive transcription of yeast ribosomal protein gene TCM1 is promoted by uncommon cis- and trans-acting elements. Mol Cell Biol. 1988 Oct;8(10):4328–4341. doi: 10.1128/mcb.8.10.4328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hodge M. R., Singh K., Cumsky M. G. Upstream activation and repression elements control transcription of the yeast COX5b gene. Mol Cell Biol. 1990 Oct;10(10):5510–5520. doi: 10.1128/mcb.10.10.5510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Lowry C. V., Cerdán M. E., Zitomer R. S. A hypoxic consensus operator and a constitutive activation region regulate the ANB1 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1990 Nov;10(11):5921–5926. doi: 10.1128/mcb.10.11.5921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lowry C. V., Lieber R. H. Negative regulation of the Saccharomyces cerevisiae ANB1 gene by heme, as mediated by the ROX1 gene product. Mol Cell Biol. 1986 Dec;6(12):4145–4148. doi: 10.1128/mcb.6.12.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lowry C. V., Zitomer R. S. Oxygen regulation of anaerobic and aerobic genes mediated by a common factor in yeast. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6129–6133. doi: 10.1073/pnas.81.19.6129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
  29. Olesen J., Hahn S., Guarente L. Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner. Cell. 1987 Dec 24;51(6):953–961. doi: 10.1016/0092-8674(87)90582-4. [DOI] [PubMed] [Google Scholar]
  30. Patterson T. E., Poyton R. O. COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini. J Biol Chem. 1986 Dec 25;261(36):17192–17197. [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., Prezant T., Guarente L. Yeast HAP1 activator binds to two upstream activation sites of different sequence. Cell. 1987 Apr 10;49(1):19–27. doi: 10.1016/0092-8674(87)90751-3. [DOI] [PubMed] [Google Scholar]
  33. Pinkham J. L., Olesen J. T., Guarente L. P. Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator. Mol Cell Biol. 1987 Feb;7(2):578–585. doi: 10.1128/mcb.7.2.578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Power S. D., Lochrie M. A., Sevarino K. A., Patterson T. E., Poyton R. O. The nuclear-coded subunits of yeast cytochrome c oxidase. I. Fractionation of the holoenzyme into chemically pure polypeptides and the identification of two new subunits using solvent extraction and reversed phase high performance liquid chromatography. J Biol Chem. 1984 May 25;259(10):6564–6570. [PubMed] [Google Scholar]
  35. Poyton R. O. Cooperative interaction between mitochondrial and nuclear genomes: cytochrome c oxidase assembly as a model. Curr Top Cell Regul. 1980;17:231–295. doi: 10.1016/b978-0-12-152817-1.50012-2. [DOI] [PubMed] [Google Scholar]
  36. Poyton R. O., Trueblood C. E., Wright R. M., Farrell L. E. Expression and function of cytochrome c oxidase subunit isologues. Modulators of cellular energy production? Ann N Y Acad Sci. 1988;550:289–307. doi: 10.1111/j.1749-6632.1988.tb35344.x. [DOI] [PubMed] [Google Scholar]
  37. Prezant T., Pfeifer K., Guarente L. Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation. Mol Cell Biol. 1987 Sep;7(9):3252–3259. doi: 10.1128/mcb.7.9.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rhode P. R., Sweder K. S., Oegema K. F., Campbell J. L. The gene encoding ARS-binding factor I is essential for the viability of yeast. Genes Dev. 1989 Dec;3(12A):1926–1939. doi: 10.1101/gad.3.12a.1926. [DOI] [PubMed] [Google Scholar]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shore D., Stillman D. J., Brand A. H., Nasmyth K. A. Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication. EMBO J. 1987 Feb;6(2):461–467. doi: 10.1002/j.1460-2075.1987.tb04776.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thorsness M., Schafer W., D'Ari L., Rine J. Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Dec;9(12):5702–5712. doi: 10.1128/mcb.9.12.5702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Trawick J. D., Rogness C., Poyton R. O. Identification of an upstream activation sequence and other cis-acting elements required for transcription of COX6 from Saccharomyces cerevisiae. Mol Cell Biol. 1989 Dec;9(12):5350–5358. doi: 10.1128/mcb.9.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Trawick J. D., Wright R. M., Poyton R. O. Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene. J Biol Chem. 1989 Apr 25;264(12):7005–7008. [PubMed] [Google Scholar]
  44. Trueblood C. E., Wright R. M., Poyton R. O. Differential regulation of the two genes encoding Saccharomyces cerevisiae cytochrome c oxidase subunit V by heme and the HAP2 and REO1 genes. Mol Cell Biol. 1988 Oct;8(10):4537–4540. doi: 10.1128/mcb.8.10.4537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vignais M. L., Woudt L. P., Wassenaar G. M., Mager W. H., Sentenac A., Planta R. J. Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes. EMBO J. 1987 May;6(5):1451–1457. doi: 10.1002/j.1460-2075.1987.tb02386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Winkler H., Adam G., Mattes E., Schanz M., Hartig A., Ruis H. Co-ordinate control of synthesis of mitochondrial and non-mitochondrial hemoproteins: a binding site for the HAP1 (CYP1) protein in the UAS region of the yeast catalase T gene (CTT1). EMBO J. 1988 Jun;7(6):1799–1804. doi: 10.1002/j.1460-2075.1988.tb03011.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Woods R. A., Sanders H. K., Briquet M., Foury F., Drysdale B. E., Mattoon J. R. Regulation of mitochondrial biogenesis: enzymatic changes in cytochrome-deficient yeast mutants requiring delta-aminolevulinic acid. J Biol Chem. 1975 Dec 10;250(23):9090–9098. [PubMed] [Google Scholar]
  48. Wright C. F., Zitomer R. S. A positive regulatory site and a negative regulatory site control the expression of the Saccharomyces cerevisiae CYC7 gene. Mol Cell Biol. 1984 Oct;4(10):2023–2030. doi: 10.1128/mcb.4.10.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wright R. M., Dircks L. K., Poyton R. O. Characterization of COX9, the nuclear gene encoding the yeast mitochondrial protein cytochrome c oxidase subunit VIIa. Subunit VIIa lacks a leader peptide and is an essential component of the holoenzyme. J Biol Chem. 1986 Dec 25;261(36):17183–17191. [PubMed] [Google Scholar]
  50. Wright R. M., Ko C., Cumsky M. G., Poyton R. O. Isolation and sequence of the structural gene for cytochrome c oxidase subunit VI from Saccharomyces cerevisiae. J Biol Chem. 1984 Dec 25;259(24):15401–15407. [PubMed] [Google Scholar]
  51. Wright R. M., Poyton R. O. Release of two Saccharomyces cerevisiae cytochrome genes, COX6 and CYC1, from glucose repression requires the SNF1 and SSN6 gene products. Mol Cell Biol. 1990 Mar;10(3):1297–1300. doi: 10.1128/mcb.10.3.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wright R. M., Rosenzweig B., Poyton R. O. Organization and expression of the COX6 genetic locus in Saccharomyces cerevisiae: multiple mRNAs with different 3' termini are transcribed from COX6 and regulated differentially. Nucleic Acids Res. 1989 Feb 11;17(3):1103–1120. doi: 10.1093/nar/17.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zagorec M., Labbe-Bois R. Negative control of yeast coproporphyrinogen oxidase synthesis by heme and oxygen. J Biol Chem. 1986 Feb 25;261(6):2506–2509. [PubMed] [Google Scholar]
  54. Zitomer R. S., Sellers J. W., McCarter D. W., Hastings G. A., Wick P., Lowry C. V. Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene. Mol Cell Biol. 1987 Jun;7(6):2212–2220. doi: 10.1128/mcb.7.6.2212. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES