Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Apr 15;25(8):1633–1640. doi: 10.1093/nar/25.8.1633

An immunoaffinity purified Schizosaccharomyces pombe TBP-containing complex directs correct initiation of the S.pombe rRNA gene promoter.

L Chen 1, A Guo 1, L Pape 1
PMCID: PMC146630  PMID: 9092673

Abstract

The multi-protein complex SL1, containing TBP, which is essential for RNA polymerase I catalyzed transcription, has been analyzed in fission yeast. It was immunopurified based on association of component subunits with epitope-tagged TBP. To enable this analysis, a strain of Schizosaccharomyces pombe was created where the only functional TBP coding sequences were those of FLAG-TBP. RNA polymerase I transcription components were fractionated from this strain and the TBP-associated polypeptides were subsequently immunopurified together with the epitope- tagged TBP. An assessment of the activity of this candidate SL1 complex was undertaken cross-species. This fission yeast TBP-containing complex displays two activities in redirecting transcriptional initiation of an S. pombe rDNA gene promoter cross-species in Saccharomyces cerevisiae transcription reactions: it both blocks an incorrect transcriptional start site at +7 and directs initiation at the correct site for S. pombe rRNA synthesis. This complex is essential for accurate initiation of the S.pombe rRNA gene: rRNA synthesis is reconstituted when this S.pombe TBP-containing complex is combined with a S.pombe fraction immunodepleted of TBP.

Full Text

The Full Text of this article is available as a PDF (169.6 KB).

Selected References

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

  1. Bateman E., Paule M. R. Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification. Cell. 1986 Nov 7;47(3):445–450. doi: 10.1016/0092-8674(86)90601-x. [DOI] [PubMed] [Google Scholar]
  2. Brun R. P., Ryan K., Sollner-Webb B. Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process. Mol Cell Biol. 1994 Jul;14(7):5010–5021. doi: 10.1128/mcb.14.7.5010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buratowski S., Zhou H. A suppressor of TBP mutations encodes an RNA polymerase III transcription factor with homology to TFIIB. Cell. 1992 Oct 16;71(2):221–230. doi: 10.1016/0092-8674(92)90351-c. [DOI] [PubMed] [Google Scholar]
  4. Choe S. Y., Schultz M. C., Reeder R. H. In vitro definition of the yeast RNA polymerase I promoter. Nucleic Acids Res. 1992 Jan 25;20(2):279–285. doi: 10.1093/nar/20.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Comai L., Tanese N., Tjian R. The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1. Cell. 1992 Mar 6;68(5):965–976. doi: 10.1016/0092-8674(92)90039-f. [DOI] [PubMed] [Google Scholar]
  6. Comai L., Zomerdijk J. C., Beckmann H., Zhou S., Admon A., Tjian R. Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits. Science. 1994 Dec 23;266(5193):1966–1972. doi: 10.1126/science.7801123. [DOI] [PubMed] [Google Scholar]
  7. Cormack B. P., Struhl K. The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells. Cell. 1992 May 15;69(4):685–696. doi: 10.1016/0092-8674(92)90232-2. [DOI] [PubMed] [Google Scholar]
  8. Eberhard D., Tora L., Egly J. M., Grummt I. A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I. Nucleic Acids Res. 1993 Sep 11;21(18):4180–4186. doi: 10.1093/nar/21.18.4180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fikes J. D., Becker D. M., Winston F., Guarente L. Striking conservation of TFIID in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Nature. 1990 Jul 19;346(6281):291–294. doi: 10.1038/346291a0. [DOI] [PubMed] [Google Scholar]
  10. Gong X., Radebaugh C. A., Geiss G. K., Simon M. N., Paule M. R. Site-directed photo-cross-linking of rRNA transcription initiation complexes. Mol Cell Biol. 1995 Sep;15(9):4956–4963. doi: 10.1128/mcb.15.9.4956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hahn S., Buratowski S., Sharp P. A., Guarente L. Isolation of the gene encoding the yeast TATA binding protein TFIID: a gene identical to the SPT15 suppressor of Ty element insertions. Cell. 1989 Sep 22;58(6):1173–1181. doi: 10.1016/0092-8674(89)90515-1. [DOI] [PubMed] [Google Scholar]
  12. Hempel W. M., Cavanaugh A. H., Hannan R. D., Taylor L., Rothblum L. I. The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor. Mol Cell Biol. 1996 Feb;16(2):557–563. doi: 10.1128/mcb.16.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hernandez N. TBP, a universal eukaryotic transcription factor? Genes Dev. 1993 Jul;7(7B):1291–1308. doi: 10.1101/gad.7.7b.1291. [DOI] [PubMed] [Google Scholar]
  14. Hoffmann A., Horikoshi M., Wang C. K., Schroeder S., Weil P. A., Roeder R. G. Cloning of the Schizosaccharomyces pombe TFIID gene reveals a strong conservation of functional domains present in Saccharomyces cerevisiae TFIID. Genes Dev. 1990 Jul;4(7):1141–1148. doi: 10.1101/gad.4.7.1141. [DOI] [PubMed] [Google Scholar]
  15. Jantzen H. M., Chow A. M., King D. S., Tjian R. Multiple domains of the RNA polymerase I activator hUBF interact with the TATA-binding protein complex hSL1 to mediate transcription. Genes Dev. 1992 Oct;6(10):1950–1963. doi: 10.1101/gad.6.10.1950. [DOI] [PubMed] [Google Scholar]
  16. Kassavetis G. A., Nguyen S. T., Kobayashi R., Kumar A., Geiduschek E. P., Pisano M. Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9786–9790. doi: 10.1073/pnas.92.21.9786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keys D. A., Lee B. S., Dodd J. A., Nguyen T. T., Vu L., Fantino E., Burson L. M., Nogi Y., Nomura M. Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex. Genes Dev. 1996 Apr 1;10(7):887–903. doi: 10.1101/gad.10.7.887. [DOI] [PubMed] [Google Scholar]
  18. Keys D. A., Vu L., Steffan J. S., Dodd J. A., Yamamoto R. T., Nogi Y., Nomura M. RRN6 and RRN7 encode subunits of a multiprotein complex essential for the initiation of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae. Genes Dev. 1994 Oct 1;8(19):2349–2362. doi: 10.1101/gad.8.19.2349. [DOI] [PubMed] [Google Scholar]
  19. Kownin P., Bateman E., Paule M. R. Eukaryotic RNA polymerase I promoter binding is directed by protein contacts with transcription initiation factor and is DNA sequence-independent. Cell. 1987 Aug 28;50(5):693–699. doi: 10.1016/0092-8674(87)90327-8. [DOI] [PubMed] [Google Scholar]
  20. Kulkens T., Riggs D. L., Heck J. D., Planta R. J., Nomura M. The yeast RNA polymerase I promoter: ribosomal DNA sequences involved in transcription initiation and complex formation in vitro. Nucleic Acids Res. 1991 Oct 11;19(19):5363–5370. doi: 10.1093/nar/19.19.5363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kwon H., Green M. R. The RNA polymerase I transcription factor, upstream binding factor, interacts directly with the TATA box-binding protein. J Biol Chem. 1994 Dec 2;269(48):30140–30146. [PubMed] [Google Scholar]
  22. Lalo D., Steffan J. S., Dodd J. A., Nomura M. RRN11 encodes the third subunit of the complex containing Rrn6p and Rrn7p that is essential for the initiation of rDNA transcription by yeast RNA polymerase I. J Biol Chem. 1996 Aug 30;271(35):21062–21067. doi: 10.1074/jbc.271.35.21062. [DOI] [PubMed] [Google Scholar]
  23. Lin C. W., Moorefield B., Payne J., Aprikian P., Mitomo K., Reeder R. H. A novel 66-kilodalton protein complexes with Rrn6, Rrn7, and TATA-binding protein to promote polymerase I transcription initiation in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Nov;16(11):6436–6443. doi: 10.1128/mcb.16.11.6436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu Z., Zhao A., Chen L., Pape L. Activated levels of rRNA synthesis in fission yeast are driven by an intergenic rDNA region positioned over 2500 nucleotides upstream of the initiation site. Nucleic Acids Res. 1997 Feb 1;25(3):659–667. doi: 10.1093/nar/25.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. López-De-León A., Librizzi M., Puglia K., Willis I. M. PCF4 encodes an RNA polymerase III transcription factor with homology to TFIIB. Cell. 1992 Oct 16;71(2):211–220. doi: 10.1016/0092-8674(92)90350-l. [DOI] [PubMed] [Google Scholar]
  26. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  27. McBryant S. J., Meier E., Leresche A., Sharp S. J., Wolf V. J., Gottesfeld J. M. TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis. Mol Cell Biol. 1996 Sep;16(9):4639–4647. doi: 10.1128/mcb.16.9.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McStay B., Hu C. H., Pikaard C. S., Reeder R. H. xUBF and Rib 1 are both required for formation of a stable polymerase I promoter complex in X. laevis. EMBO J. 1991 Aug;10(8):2297–2303. doi: 10.1002/j.1460-2075.1991.tb07766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meyers R. E., Sharp P. A. TATA-binding protein and associated factors in polymerase II and polymerase III transcription. Mol Cell Biol. 1993 Dec;13(12):7953–7960. doi: 10.1128/mcb.13.12.7953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mishima Y., Financsek I., Kominami R., Muramatsu M. Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-dependent initiation factor. Nucleic Acids Res. 1982 Nov 11;10(21):6659–6670. doi: 10.1093/nar/10.21.6659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  32. Pape L. K., Windle J. J., Sollner-Webb B. Half helical turn spacing changes convert a frog into a mouse rDNA promoter: a distant upstream domain determines the helix face of the initiation site. Genes Dev. 1990 Jan;4(1):52–62. doi: 10.1101/gad.4.1.52. [DOI] [PubMed] [Google Scholar]
  33. Poon D., Bai Y., Campbell A. M., Bjorklund S., Kim Y. J., Zhou S., Kornberg R. D., Weil P. A. Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8224–8228. doi: 10.1073/pnas.92.18.8224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Radebaugh C. A., Matthews J. L., Geiss G. K., Liu F., Wong J. M., Bateman E., Camier S., Sentenac A., Paule M. R. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors. Mol Cell Biol. 1994 Jan;14(1):597–605. doi: 10.1128/mcb.14.1.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Riggs D. L., Peterson C. L., Wickham J. Q., Miller L. M., Clarke E. M., Crowell J. A., Sergere J. C. Characterization of the components of reconstituted Saccharomyces cerevisiae RNA polymerase I transcription complexes. J Biol Chem. 1995 Mar 17;270(11):6205–6210. doi: 10.1074/jbc.270.11.6205. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Rudloff U., Eberhard D., Tora L., Stunnenberg H., Grummt I. TBP-associated factors interact with DNA and govern species specificity of RNA polymerase I transcription. EMBO J. 1994 Jun 1;13(11):2611–2616. doi: 10.1002/j.1460-2075.1994.tb06551.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Schnapp A., Grummt I. Transcription complex formation at the mouse rDNA promoter involves the stepwise association of four transcription factors and RNA polymerase I. J Biol Chem. 1991 Dec 25;266(36):24588–24595. [PubMed] [Google Scholar]
  41. Schnapp G., Schnapp A., Rosenbauer H., Grummt I. TIF-IC, a factor involved in both transcription initiation and elongation of RNA polymerase I. EMBO J. 1994 Sep 1;13(17):4028–4035. doi: 10.1002/j.1460-2075.1994.tb06719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schultz M. C., Choe S. Y., Reeder R. H. Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1004–1008. doi: 10.1073/pnas.88.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schultz M. C., Reeder R. H., Hahn S. Variants of the TATA-binding protein can distinguish subsets of RNA polymerase I, II, and III promoters. Cell. 1992 May 15;69(4):697–702. doi: 10.1016/0092-8674(92)90233-3. [DOI] [PubMed] [Google Scholar]
  44. Smith S. D., Oriahi E., Lowe D., Yang-Yen H. F., O'Mahony D., Rose K., Chen K., Rothblum L. I. Characterization of factors that direct transcription of rat ribosomal DNA. Mol Cell Biol. 1990 Jun;10(6):3105–3116. doi: 10.1128/mcb.10.6.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sollner-Webb B., Tower J. Transcription of cloned eukaryotic ribosomal RNA genes. Annu Rev Biochem. 1986;55:801–830. doi: 10.1146/annurev.bi.55.070186.004101. [DOI] [PubMed] [Google Scholar]
  46. Steffan J. S., Keys D. A., Dodd J. A., Nomura M. The role of TBP in rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae: TBP is required for upstream activation factor-dependent recruitment of core factor. Genes Dev. 1996 Oct 15;10(20):2551–2563. doi: 10.1101/gad.10.20.2551. [DOI] [PubMed] [Google Scholar]
  47. Tanese N., Tjian R. Coactivators and TAFs: a new class of eukaryotic transcription factors that connect activators to the basal machinery. Cold Spring Harb Symp Quant Biol. 1993;58:179–185. doi: 10.1101/sqb.1993.058.01.022. [DOI] [PubMed] [Google Scholar]
  48. Tower J., Sollner-Webb B. Transcription of mouse rDNA is regulated by an activated subform of RNA polymerase I. Cell. 1987 Sep 11;50(6):873–883. doi: 10.1016/0092-8674(87)90514-9. [DOI] [PubMed] [Google Scholar]
  49. Yamamoto R. T., Nogi Y., Dodd J. A., Nomura M. RRN3 gene of Saccharomyces cerevisiae encodes an essential RNA polymerase I transcription factor which interacts with the polymerase independently of DNA template. EMBO J. 1996 Aug 1;15(15):3964–3973. [PMC free article] [PubMed] [Google Scholar]
  50. Yang Q., Radebaugh C. A., Kubaska W., Geiss G. K., Paule M. R. Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions. Nucleic Acids Res. 1995 Nov 11;23(21):4345–4352. doi: 10.1093/nar/23.21.4345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou Q., Boyer T. G., Berk A. J. Factors (TAFs) required for activated transcription interact with TATA box-binding protein conserved core domain. Genes Dev. 1993 Feb;7(2):180–187. doi: 10.1101/gad.7.2.180. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Zomerdijk J. C., Beckmann H., Comai L., Tjian R. Assembly of transcriptionally active RNA polymerase I initiation factor SL1 from recombinant subunits. Science. 1994 Dec 23;266(5193):2015–2018. doi: 10.1126/science.7801130. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES