Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Nov;11(11):5648–5659. doi: 10.1128/mcb.11.11.5648

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.

F J McNally 1, J Rine 1
PMCID: PMC361936  PMID: 1922068

Abstract

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

Full text

PDF
5656

Images in this article

5652Image
on p.5652
5653Image
on p.5653
5654Image
on p.5654
5654Image
on p.5654

Selected References

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

  1. Abraham J., Nasmyth K. A., Strathern J. N., Klar A. J., Hicks J. B. Regulation of mating-type information in yeast. Negative control requiring sequences both 5' and 3' to the regulated region. J Mol Biol. 1984 Jul 5;176(3):307–331. doi: 10.1016/0022-2836(84)90492-3. [DOI] [PubMed] [Google Scholar]
  2. Astell C. R., Ahlstrom-Jonasson L., Smith M., Tatchell K., Nasmyth K. A., Hall B. D. The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae. Cell. 1981 Nov;27(1 Pt 2):15–23. doi: 10.1016/0092-8674(81)90356-1. [DOI] [PubMed] [Google Scholar]
  3. Banerji J., Rusconi S., Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981 Dec;27(2 Pt 1):299–308. doi: 10.1016/0092-8674(81)90413-x. [DOI] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bouton A. H., Smith M. M. Fine-structure analysis of the DNA sequence requirements for autonomous replication of Saccharomyces cerevisiae plasmids. Mol Cell Biol. 1986 Jul;6(7):2354–2363. doi: 10.1128/mcb.6.7.2354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brand A. H., Breeden L., Abraham J., Sternglanz R., Nasmyth K. Characterization of a "silencer" in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer. Cell. 1985 May;41(1):41–48. doi: 10.1016/0092-8674(85)90059-5. [DOI] [PubMed] [Google Scholar]
  7. Brand A. H., Micklem G., Nasmyth K. A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell. 1987 Dec 4;51(5):709–719. doi: 10.1016/0092-8674(87)90094-8. [DOI] [PubMed] [Google Scholar]
  8. Brewer B. J., Fangman W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. doi: 10.1016/0092-8674(87)90642-8. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Diffley J. F., Stillman B. Purification of a yeast protein that binds to origins of DNA replication and a transcriptional silencer. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2120–2124. doi: 10.1073/pnas.85.7.2120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Diffley J. F., Stillman B. Similarity between the transcriptional silencer binding proteins ABF1 and RAP1. Science. 1989 Nov 24;246(4933):1034–1038. doi: 10.1126/science.2511628. [DOI] [PubMed] [Google Scholar]
  14. Emr S. D., Vassarotti A., Garrett J., Geller B. L., Takeda M., Douglas M. G. The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol. 1986 Feb;102(2):523–533. doi: 10.1083/jcb.102.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fangman W. L., Hice R. H., Chlebowicz-Sledziewska E. ARS replication during the yeast S phase. Cell. 1983 Mar;32(3):831–838. doi: 10.1016/0092-8674(83)90069-7. [DOI] [PubMed] [Google Scholar]
  16. Giesman D., Best L., Tatchell K. The role of RAP1 in the regulation of the MAT alpha locus. Mol Cell Biol. 1991 Feb;11(2):1069–1079. doi: 10.1128/mcb.11.2.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Herskowitz I. Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiol Rev. 1988 Dec;52(4):536–553. doi: 10.1128/mr.52.4.536-553.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hofmann J. F., Gasser S. M. Identification and purification of a protein that binds the yeast ARS consensus sequence. Cell. 1991 Mar 8;64(5):951–960. doi: 10.1016/0092-8674(91)90319-t. [DOI] [PubMed] [Google Scholar]
  19. Holm C., Meeks-Wagner D. W., Fangman W. L., Botstein D. A rapid, efficient method for isolating DNA from yeast. Gene. 1986;42(2):169–173. doi: 10.1016/0378-1119(86)90293-3. [DOI] [PubMed] [Google Scholar]
  20. Huet J., Cottrelle P., Cool M., Vignais M. L., Thiele D., Marck C., Buhler J. M., Sentenac A., Fromageot P. A general upstream binding factor for genes of the yeast translational apparatus. EMBO J. 1985 Dec 16;4(13A):3539–3547. doi: 10.1002/j.1460-2075.1985.tb04114.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kassir Y., Hicks J. B., Herskowitz I. SAD mutation of Saccharomyces cerevisiae is an extra a cassette. Mol Cell Biol. 1983 May;3(5):871–880. doi: 10.1128/mcb.3.5.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kayne P. S., Kim U. J., Han M., Mullen J. R., Yoshizaki F., Grunstein M. Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast. Cell. 1988 Oct 7;55(1):27–39. doi: 10.1016/0092-8674(88)90006-2. [DOI] [PubMed] [Google Scholar]
  23. Kimmerly W. J., Rine J. Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes. Mol Cell Biol. 1987 Dec;7(12):4225–4237. doi: 10.1128/mcb.7.12.4225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kimmerly W., Buchman A., Kornberg R., Rine J. Roles of two DNA-binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer. EMBO J. 1988 Jul;7(7):2241–2253. doi: 10.1002/j.1460-2075.1988.tb03064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kipling D., Kearsey S. E. Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA. Mol Cell Biol. 1990 Jan;10(1):265–272. doi: 10.1128/mcb.10.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klar A. J., Strathern J. N., Hicks J. B. A position-effect control for gene transposition: state of expression of yeast mating-type genes affects their ability to switch. Cell. 1981 Aug;25(2):517–524. doi: 10.1016/0092-8674(81)90070-2. [DOI] [PubMed] [Google Scholar]
  27. Kurtz S., Shore D. RAP1 protein activates and silences transcription of mating-type genes in yeast. Genes Dev. 1991 Apr;5(4):616–628. doi: 10.1101/gad.5.4.616. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Miller A. M., Nasmyth K. A. Role of DNA replication in the repression of silent mating type loci in yeast. Nature. 1984 Nov 15;312(5991):247–251. doi: 10.1038/312247a0. [DOI] [PubMed] [Google Scholar]
  30. Miller A. M. The yeast MATa1 gene contains two introns. EMBO J. 1984 May;3(5):1061–1065. doi: 10.1002/j.1460-2075.1984.tb01927.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murray A. W., Szostak J. W. Pedigree analysis of plasmid segregation in yeast. Cell. 1983 Oct;34(3):961–970. doi: 10.1016/0092-8674(83)90553-6. [DOI] [PubMed] [Google Scholar]
  32. Nasmyth K. A., Tatchell K., Hall B. D., Astell C., Smith M. A position effect in the control of transcription at yeast mating type loci. Nature. 1981 Jan 22;289(5795):244–250. doi: 10.1038/289244a0. [DOI] [PubMed] [Google Scholar]
  33. Palzkill T. G., Newlon C. S. A yeast replication origin consists of multiple copies of a small conserved sequence. Cell. 1988 May 6;53(3):441–450. doi: 10.1016/0092-8674(88)90164-x. [DOI] [PubMed] [Google Scholar]
  34. Parent S. A., Fenimore C. M., Bostian K. A. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast. 1985 Dec;1(2):83–138. doi: 10.1002/yea.320010202. [DOI] [PubMed] [Google Scholar]
  35. Pillus L., Rine J. Epigenetic inheritance of transcriptional states in S. cerevisiae. Cell. 1989 Nov 17;59(4):637–647. doi: 10.1016/0092-8674(89)90009-3. [DOI] [PubMed] [Google Scholar]
  36. Rine J., Herskowitz I. Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. Genetics. 1987 May;116(1):9–22. doi: 10.1093/genetics/116.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rine J., Strathern J. N., Hicks J. B., Herskowitz I. A suppressor of mating-type locus mutations in Saccharomyces cerevisiae: evidence for and identification of cryptic mating-type loci. Genetics. 1979 Dec;93(4):877–901. doi: 10.1093/genetics/93.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [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. Schnell R., Rine J. A position effect on the expression of a tRNA gene mediated by the SIR genes in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Feb;6(2):494–501. doi: 10.1128/mcb.6.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Shore D., Squire M., Nasmyth K. A. Characterization of two genes required for the position-effect control of yeast mating-type genes. EMBO J. 1984 Dec 1;3(12):2817–2823. doi: 10.1002/j.1460-2075.1984.tb02214.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Siliciano P. G., Tatchell K. Transcription and regulatory signals at the mating type locus in yeast. Cell. 1984 Jul;37(3):969–978. doi: 10.1016/0092-8674(84)90431-8. [DOI] [PubMed] [Google Scholar]
  46. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  47. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Van Houten J. V., Newlon C. S. Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III. Mol Cell Biol. 1990 Aug;10(8):3917–3925. doi: 10.1128/mcb.10.8.3917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  50. Zweifel S. G., Fangman W. L. Creation of ARS activity in yeast through iteration of non-functional sequences. Yeast. 1990 May-Jun;6(3):179–186. doi: 10.1002/yea.320060302. [DOI] [PubMed] [Google Scholar]

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

RESOURCES