Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Apr;16(4):1641–1648. doi: 10.1128/mcb.16.4.1641

Transcriptional corepression in vitro: a Mot1p-associated form of TATA-binding protein is required for repression by Leu3p.

P A Wade 1, J A Jaehning 1
PMCID: PMC231150  PMID: 8657139

Abstract

Signals from transcriptional activators to the general mRNA transcription apparatus are communicated by factors associated with RNA polymerase II or the TATA-binding protein (TBP). Currently, little is known about how gene-specific transcription repressors communicate with RNA polymerase II. We have analyzed the requirements for repression by the saccharomyces cerevisiae Leu3 protein (Leu3p) in a reconstituted transcription system. We have identified a complex form of TBP which is required for communication of the repressing signal. This TFIID-like complex contains a known TBP-associated protein, Mot1p, which has been implicated in the repression of a subset of yeast genes by genetic analysis. Leu3p-dependent repression can be reconstituted with purified Mot1p and recombinant TBP. In addition, a mutation in the Mot1 gene leads to partial derepression of the Leu3p-dependent LEU2 promoter. These in vivo and in vitro observations define a role for Mot1p as a transcriptional corepressor.

Full Text

The Full Text of this article is available as a PDF (426.8 KB).

Selected References

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

  1. Andreadis A., Hsu Y. P., Kohlhaw G. B., Schimmel P. Nucleotide sequence of yeast LEU2 shows 5'-noncoding region has sequences cognate to leucine. Cell. 1982 Dec;31(2 Pt 1):319–325. doi: 10.1016/0092-8674(82)90125-8. [DOI] [PubMed] [Google Scholar]
  2. Auble D. T., Hahn S. An ATP-dependent inhibitor of TBP binding to DNA. Genes Dev. 1993 May;7(5):844–856. doi: 10.1101/gad.7.5.844. [DOI] [PubMed] [Google Scholar]
  3. Auble D. T., Hansen K. E., Mueller C. G., Lane W. S., Thorner J., Hahn S. Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 1994 Aug 15;8(16):1920–1934. doi: 10.1101/gad.8.16.1920. [DOI] [PubMed] [Google Scholar]
  4. Austin R. J., Biggin M. D. A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking. Mol Cell Biol. 1995 Sep;15(9):4683–4693. doi: 10.1128/mcb.15.9.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brisco P. R., Kohlhaw G. B. Regulation of yeast LEU2. Total deletion of regulatory gene LEU3 unmasks GCN4-dependent basal level expression of LEU2. J Biol Chem. 1990 Jul 15;265(20):11667–11675. [PubMed] [Google Scholar]
  6. Chen J. L., Attardi L. D., Verrijzer C. P., Yokomori K., Tjian R. Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators. Cell. 1994 Oct 7;79(1):93–105. doi: 10.1016/0092-8674(94)90403-0. [DOI] [PubMed] [Google Scholar]
  7. Cortes P., Flores O., Reinberg D. Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ. Mol Cell Biol. 1992 Jan;12(1):413–421. doi: 10.1128/mcb.12.1.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis J. L., Kunisawa R., Thorner J. A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1992 Apr;12(4):1879–1892. doi: 10.1128/mcb.12.4.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dynlacht B. D., Hoey T., Tjian R. Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation. Cell. 1991 Aug 9;66(3):563–576. doi: 10.1016/0092-8674(81)90019-2. [DOI] [PubMed] [Google Scholar]
  10. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Friden P., Schimmel P. LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence. Mol Cell Biol. 1988 Jul;8(7):2690–2697. doi: 10.1128/mcb.8.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ge H., Roeder R. G. The high mobility group protein HMG1 can reversibly inhibit class II gene transcription by interaction with the TATA-binding protein. J Biol Chem. 1994 Jun 24;269(25):17136–17140. [PubMed] [Google Scholar]
  13. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hisatake K., Hasegawa S., Takada R., Nakatani Y., Horikoshi M., Roeder R. G. The p250 subunit of native TATA box-binding factor TFIID is the cell-cycle regulatory protein CCG1. Nature. 1993 Mar 11;362(6416):179–181. doi: 10.1038/362179a0. [DOI] [PubMed] [Google Scholar]
  15. Hu Y., Cooper T. G., Kohlhaw G. B. The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation. Mol Cell Biol. 1995 Jan;15(1):52–57. doi: 10.1128/mcb.15.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacq X., Brou C., Lutz Y., Davidson I., Chambon P., Tora L. Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor. Cell. 1994 Oct 7;79(1):107–117. doi: 10.1016/0092-8674(94)90404-9. [DOI] [PubMed] [Google Scholar]
  17. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  18. Kokubo T., Gong D. W., Roeder R. G., Horikoshi M., Nakatani Y. The Drosophila 110-kDa transcription factor TFIID subunit directly interacts with the N-terminal region of the 230-kDa subunit. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5896–5900. doi: 10.1073/pnas.90.13.5896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kokubo T., Gong D. W., Yamashita S., Horikoshi M., Roeder R. G., Nakatani Y. Drosophila 230-kD TFIID subunit, a functional homolog of the human cell cycle gene product, negatively regulates DNA binding of the TATA box-binding subunit of TFIID. Genes Dev. 1993 Jun;7(6):1033–1046. doi: 10.1101/gad.7.6.1033. [DOI] [PubMed] [Google Scholar]
  20. Koleske A. J., Young R. A. An RNA polymerase II holoenzyme responsive to activators. Nature. 1994 Mar 31;368(6470):466–469. doi: 10.1038/368466a0. [DOI] [PubMed] [Google Scholar]
  21. Komachi K., Redd M. J., Johnson A. D. The WD repeats of Tup1 interact with the homeo domain protein alpha 2. Genes Dev. 1994 Dec 1;8(23):2857–2867. doi: 10.1101/gad.8.23.2857. [DOI] [PubMed] [Google Scholar]
  22. Kuchin S., Yeghiayan P., Carlson M. Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):4006–4010. doi: 10.1073/pnas.92.9.4006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Paranjape S. M., Kamakaka R. T., Kadonaga J. T. Role of chromatin structure in the regulation of transcription by RNA polymerase II. Annu Rev Biochem. 1994;63:265–297. doi: 10.1146/annurev.bi.63.070194.001405. [DOI] [PubMed] [Google Scholar]
  24. Parthun M. R., Mangus D. A., Jaehning J. A. The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding. Mol Cell Biol. 1992 Dec;12(12):5683–5689. doi: 10.1128/mcb.12.12.5683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Poon D., Campbell A. M., Bai Y., Weil P. A. Yeast Taf170 is encoded by MOT1 and exists in a TATA box-binding protein (TBP)-TBP-associated factor complex distinct from transcription factor IID. J Biol Chem. 1994 Sep 16;269(37):23135–23140. [PubMed] [Google Scholar]
  26. Poon D., Weil P. A. Immunopurification of yeast TATA-binding protein and associated factors. Presence of transcription factor IIIB transcriptional activity. J Biol Chem. 1993 Jul 25;268(21):15325–15328. [PubMed] [Google Scholar]
  27. Pugh B. F., Tjian R. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes Dev. 1991 Nov;5(11):1935–1945. doi: 10.1101/gad.5.11.1935. [DOI] [PubMed] [Google Scholar]
  28. Reddy P., Hahn S. Dominant negative mutations in yeast TFIID define a bipartite DNA-binding region. Cell. 1991 Apr 19;65(2):349–357. doi: 10.1016/0092-8674(91)90168-x. [DOI] [PubMed] [Google Scholar]
  29. Reese J. C., Apone L., Walker S. S., Griffin L. A., Green M. R. Yeast TAFIIS in a multisubunit complex required for activated transcription. Nature. 1994 Oct 6;371(6497):523–527. doi: 10.1038/371523a0. [DOI] [PubMed] [Google Scholar]
  30. Remboutsika E., Kohlhaw G. B. Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach. Mol Cell Biol. 1994 Aug;14(8):5547–5557. doi: 10.1128/mcb.14.8.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ronne H. Glucose repression in fungi. Trends Genet. 1995 Jan;11(1):12–17. doi: 10.1016/s0168-9525(00)88980-5. [DOI] [PubMed] [Google Scholar]
  32. Ruppert S., Wang E. H., Tjian R. Cloning and expression of human TAFII250: a TBP-associated factor implicated in cell-cycle regulation. Nature. 1993 Mar 11;362(6416):175–179. doi: 10.1038/362175a0. [DOI] [PubMed] [Google Scholar]
  33. Sauer F., Fondell J. D., Ohkuma Y., Roeder R. G., Jäckle H. Control of transcription by Krüppel through interactions with TFIIB and TFIIE beta. Nature. 1995 May 11;375(6527):162–164. doi: 10.1038/375162a0. [DOI] [PubMed] [Google Scholar]
  34. Sayre M. H., Tschochner H., Kornberg R. D. Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae. J Biol Chem. 1992 Nov 15;267(32):23376–23382. [PubMed] [Google Scholar]
  35. Sikorski R. S., Boguski M. S., Goebl M., Hieter P. A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell. 1990 Jan 26;60(2):307–317. doi: 10.1016/0092-8674(90)90745-z. [DOI] [PubMed] [Google Scholar]
  36. Sze J. Y., Kohlhaw G. B. Purification and structural characterization of transcriptional regulator Leu3 of yeast. J Biol Chem. 1993 Feb 5;268(4):2505–2512. [PubMed] [Google Scholar]
  37. Sze J. Y., Remboutsika E., Kohlhaw G. B. Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions. Mol Cell Biol. 1993 Sep;13(9):5702–5709. doi: 10.1128/mcb.13.9.5702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sze J. Y., Woontner M., Jaehning J. A., Kohlhaw G. B. In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate. Science. 1992 Nov 13;258(5085):1143–1145. doi: 10.1126/science.1439822. [DOI] [PubMed] [Google Scholar]
  39. Tjian R., Maniatis T. Transcriptional activation: a complex puzzle with few easy pieces. Cell. 1994 Apr 8;77(1):5–8. doi: 10.1016/0092-8674(94)90227-5. [DOI] [PubMed] [Google Scholar]
  40. Tsai M. J., O'Malley B. W. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem. 1994;63:451–486. doi: 10.1146/annurev.bi.63.070194.002315. [DOI] [PubMed] [Google Scholar]
  41. Tzamarias D., Struhl K. Distinct TPR motifs of Cyc8 are involved in recruiting the Cyc8-Tup1 corepressor complex to differentially regulated promoters. Genes Dev. 1995 Apr 1;9(7):821–831. doi: 10.1101/gad.9.7.821. [DOI] [PubMed] [Google Scholar]
  42. Um M., Li C., Manley J. L. The transcriptional repressor even-skipped interacts directly with TATA-binding protein. Mol Cell Biol. 1995 Sep;15(9):5007–5016. doi: 10.1128/mcb.15.9.5007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Verrijzer C. P., Yokomori K., Chen J. L., Tjian R. Drosophila TAFII150: similarity to yeast gene TSM-1 and specific binding to core promoter DNA. Science. 1994 May 13;264(5161):933–941. doi: 10.1126/science.8178153. [DOI] [PubMed] [Google Scholar]
  44. Wade P. A., Jaehning J. A. Isolation of yeast transcription factor IIA using a functional transcription assay. Protein Expr Purif. 1994 Dec;5(6):577–582. doi: 10.1006/prep.1994.1079. [DOI] [PubMed] [Google Scholar]
  45. Wade P. A., Shaffer S. D., Jaehning J. A. Resolution of transcription factors from a transcriptionally active whole-cell extract from yeast: purification of TFIIB, TBP, and RNA polymerase IIa. Protein Expr Purif. 1993 Aug;4(4):290–297. doi: 10.1006/prep.1993.1037. [DOI] [PubMed] [Google Scholar]
  46. Wahi M., Johnson A. D. Identification of genes required for alpha 2 repression in Saccharomyces cerevisiae. Genetics. 1995 May;140(1):79–90. doi: 10.1093/genetics/140.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weintraub S. J., Chow K. N., Luo R. X., Zhang S. H., He S., Dean D. C. Mechanism of active transcriptional repression by the retinoblastoma protein. Nature. 1995 Jun 29;375(6534):812–815. doi: 10.1038/375812a0. [DOI] [PubMed] [Google Scholar]
  48. Weinzierl R. O., Dynlacht B. D., Tjian R. Largest subunit of Drosophila transcription factor IID directs assembly of a complex containing TBP and a coactivator. Nature. 1993 Apr 8;362(6420):511–517. doi: 10.1038/362511a0. [DOI] [PubMed] [Google Scholar]
  49. Winkley C. S., Keller M. J., Jaehning J. A. A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae. J Biol Chem. 1985 Nov 15;260(26):14214–14223. [PubMed] [Google Scholar]
  50. Woontner M., Wade P. A., Bonner J., Jaehning J. A. Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae. Mol Cell Biol. 1991 Sep;11(9):4555–4560. doi: 10.1128/mcb.11.9.4555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou Q., Lieberman P. M., Boyer T. G., Berk A. J. Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter. Genes Dev. 1992 Oct;6(10):1964–1974. doi: 10.1101/gad.6.10.1964. [DOI] [PubMed] [Google Scholar]
  52. Zitomer R. S., Lowry C. V. Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microbiol Rev. 1992 Mar;56(1):1–11. doi: 10.1128/mr.56.1.1-11.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES