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. 1994 Jan 1;13(1):190–199. doi: 10.1002/j.1460-2075.1994.tb06248.x

The HMG box-containing nucleolar transcription factor UBF interacts with a specific subunit of RNA polymerase I.

G Schnapp 1, F Santori 1, C Carles 1, M Riva 1, I Grummt 1
PMCID: PMC394792  PMID: 8306961

Abstract

The mammalian transcription activator protein UBF contains five tandemly repeated HMG homology domains which are required for DNA binding. We have used highly purified RNA polymerase I (Pol I) and upstream binding factor (UBF) and investigated whether these two proteins interact in solution. We show by a variety of different experimental approaches, such as immunoprecipitation, glycerol gradient sedimentation, affinity chromatography and protein blotting, that UBF physically associates with Pol I. Mutational analysis reveals that the HMG boxes play an important role in this specific interaction. UBF binds to mouse and yeast Pol I, demonstrating that the interaction of UBF with Pol I has been conserved during evolution. Interestingly, in both species one Pol I-specific subunit (34.5 kDa in yeast and 62 kDa in mouse) was recognized by UBF. No specific interaction was observed with Pol II. Unexpectedly, UBF was found to associate also with a unique subunit of yeast Pol III. This apparent specific interaction of UBF with the two classes of RNA polymerases may reflect functionally important interactions of HMG box-containing transcription factors with the transcriptional apparatus.

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Selected References

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

  1. Annweiler A., Hipskind R. A., Wirth T. A strategy for efficient in vitro translation of cDNAs using the rabbit beta-globin leader sequence. Nucleic Acids Res. 1991 Jul 11;19(13):3750–3750. doi: 10.1093/nar/19.13.3750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bachvarov D., Moss T. The RNA polymerase I transcription factor xUBF contains 5 tandemly repeated HMG homology boxes. Nucleic Acids Res. 1991 May 11;19(9):2331–2335. doi: 10.1093/nar/19.9.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell S. P., Jantzen H. M., Tjian R. Assembly of alternative multiprotein complexes directs rRNA promoter selectivity. Genes Dev. 1990 Jun;4(6):943–954. doi: 10.1101/gad.4.6.943. [DOI] [PubMed] [Google Scholar]
  4. Bell S. P., Learned R. M., Jantzen H. M., Tjian R. Functional cooperativity between transcription factors UBF1 and SL1 mediates human ribosomal RNA synthesis. Science. 1988 Sep 2;241(4870):1192–1197. doi: 10.1126/science.3413483. [DOI] [PubMed] [Google Scholar]
  5. Bell S. P., Pikaard C. S., Reeder R. H., Tjian R. Molecular mechanisms governing species-specific transcription of ribosomal RNA. Cell. 1989 Nov 3;59(3):489–497. doi: 10.1016/0092-8674(89)90032-9. [DOI] [PubMed] [Google Scholar]
  6. Bianchi M. E., Falciola L., Ferrari S., Lilley D. M. The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins. EMBO J. 1992 Mar;11(3):1055–1063. doi: 10.1002/j.1460-2075.1992.tb05144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buhler J. M., Sentenac A., Fromageot P. Isolation, structure, and general properties of yeast ribonucleic acid polymerase A (or I). J Biol Chem. 1974 Sep 25;249(18):5963–5970. [PubMed] [Google Scholar]
  8. 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]
  9. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Giese K., Cox J., Grosschedl R. The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell. 1992 Apr 3;69(1):185–195. doi: 10.1016/0092-8674(92)90129-z. [DOI] [PubMed] [Google Scholar]
  12. Gissinger F., Chambon P. Subunit SA3 is not mandatory for the activity of calf thymus DNA-dependent RNA polymerase AI. FEBS Lett. 1975 Oct 15;58(1):53–56. doi: 10.1016/0014-5793(75)80224-9. [DOI] [PubMed] [Google Scholar]
  13. Grummt I., Roth E., Paule M. R. Ribosomal RNA transcription in vitro is species specific. Nature. 1982 Mar 11;296(5853):173–174. doi: 10.1038/296173a0. [DOI] [PubMed] [Google Scholar]
  14. Hisatake K., Nishimura T., Maeda Y., Hanada K., Song C. Z., Muramatsu M. Cloning and structural analysis of cDNA and the gene for mouse transcription factor UBF. Nucleic Acids Res. 1991 Sep 11;19(17):4631–4637. doi: 10.1093/nar/19.17.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoffmann A., Roeder R. G. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. doi: 10.1093/nar/19.22.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huet J., Buhler J. M., Sentenac A., Fromageot P. Dissociation of two polypeptide chains from yeast RNA polymerase A. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3034–3038. doi: 10.1073/pnas.72.8.3034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Igarashi K., Hanamura A., Makino K., Aiba H., Aiba H., Mizuno T., Nakata A., Ishihama A. Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8958–8962. doi: 10.1073/pnas.88.20.8958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jantzen H. M., Admon A., Bell S. P., Tjian R. Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins. Nature. 1990 Apr 26;344(6269):830–836. doi: 10.1038/344830a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kedinger C., Gissinger F., Chambon P. Animal DNA-dependent RNA polymerases. Molecular structures and immunological properties of calf-thymus enzyme AI and of calf-thymus and rat-liver enzymes B. Eur J Biochem. 1974 May 15;44(2):421–436. doi: 10.1111/j.1432-1033.1974.tb03500.x. [DOI] [PubMed] [Google Scholar]
  21. Kellas B. L., Austoker J. L., Beebee T. J., Butterworth P. H. Forms AI and AII DNA-dependent RNA polymerases as components of two defined pools of polymerase activity in mammalian cells. Eur J Biochem. 1977 Feb;72(3):583–594. doi: 10.1111/j.1432-1033.1977.tb11281.x. [DOI] [PubMed] [Google Scholar]
  22. Kuhn A., Grummt I. Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7340–7344. doi: 10.1073/pnas.89.16.7340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kuhn A., Stefanovsky V., Grummt I. The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription. Nucleic Acids Res. 1993 May 11;21(9):2057–2063. doi: 10.1093/nar/21.9.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Learned R. M., Cordes S., Tjian R. Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I. Mol Cell Biol. 1985 Jun;5(6):1358–1369. doi: 10.1128/mcb.5.6.1358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Learned R. M., Learned T. K., Haltiner M. M., Tjian R. T. Human rRNA transcription is modulated by the coordinate binding of two factors to an upstream control element. Cell. 1986 Jun 20;45(6):847–857. doi: 10.1016/0092-8674(86)90559-3. [DOI] [PubMed] [Google Scholar]
  26. Matsui T., Onishi T., Muramatsu M. Nucleolar DNA-dependent RNA polymerase from rat liver. 1. Purification and subunit structure. Eur J Biochem. 1976 Dec 11;71(2):351–360. doi: 10.1111/j.1432-1033.1976.tb11121.x. [DOI] [PubMed] [Google Scholar]
  27. McStay B., Frazier M. W., Reeder R. H. xUBF contains a novel dimerization domain essential for RNA polymerase I transcription. Genes Dev. 1991 Nov;5(11):1957–1968. doi: 10.1101/gad.5.11.1957. [DOI] [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. Miesfeld R., Arnheim N. Species-specific rDNA transcription is due to promoter-specific binding factors. Mol Cell Biol. 1984 Feb;4(2):221–227. doi: 10.1128/mcb.4.2.221. [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. O'Mahony D. J., Rothblum L. I. Identification of two forms of the RNA polymerase I transcription factor UBF. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3180–3184. doi: 10.1073/pnas.88.8.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Mahony D. J., Smith S. D., Xie W., Rothblum L. I. Analysis of the phosphorylation, DNA-binding and dimerization properties of the RNA polymerase I transcription factors UBF1 and UBF2. Nucleic Acids Res. 1992 Mar 25;20(6):1301–1308. doi: 10.1093/nar/20.6.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Onishi T., Berglund C., Reeder R. H. On the mechanism of nucleolar dominance in mouse-human somatic cell hybrids. Proc Natl Acad Sci U S A. 1984 Jan;81(2):484–487. doi: 10.1073/pnas.81.2.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Parisi M. A., Clayton D. A. Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. Science. 1991 May 17;252(5008):965–969. doi: 10.1126/science.2035027. [DOI] [PubMed] [Google Scholar]
  35. Pikaard C. S., McStay B., Schultz M. C., Bell S. P., Reeder R. H. The Xenopus ribosomal gene enhancers bind an essential polymerase I transcription factor, xUBF. Genes Dev. 1989 Nov;3(11):1779–1788. doi: 10.1101/gad.3.11.1779. [DOI] [PubMed] [Google Scholar]
  36. Pikaard C. S., Pape L. K., Henderson S. L., Ryan K., Paalman M. H., Lopata M. A., Reeder R. H., Sollner-Webb B. Enhancers for RNA polymerase I in mouse ribosomal DNA. Mol Cell Biol. 1990 Sep;10(9):4816–4825. doi: 10.1128/mcb.10.9.4816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pikaard C. S., Smith S. D., Reeder R. H., Rothblum L. rUBF, an RNA polymerase I transcription factor from rats, produces DNase I footprints identical to those produced by xUBF, its homolog from frogs. Mol Cell Biol. 1990 Jul;10(7):3810–3812. doi: 10.1128/mcb.10.7.3810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reimer G., Rose K. M., Scheer U., Tan E. M. Autoantibody to RNA polymerase I in scleroderma sera. J Clin Invest. 1987 Jan;79(1):65–72. doi: 10.1172/JCI112809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rodrigo R. M., Rendón M. C., Torreblanca J., García-Herdugo G., Moreno F. J. Characterization and immunolocalization of RNA polymerase I transcription factor UBF with anti-NOR serum in protozoa, higher plant and vertebrate cells. J Cell Sci. 1992 Dec;103(Pt 4):1053–1063. doi: 10.1242/jcs.103.4.1053. [DOI] [PubMed] [Google Scholar]
  40. Rose K. M., Stetler D. A., Jacob S. T. Protein kinase activity of RNA polymerase I purified from a rat hepatoma: probable function of Mr 42,000 and 24,600 polypeptides. Proc Natl Acad Sci U S A. 1981 May;78(5):2833–2837. doi: 10.1073/pnas.78.5.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roussel P., André C., Masson C., Géraud G., Hernandez-Verdun D. Localization of the RNA polymerase I transcription factor hUBF during the cell cycle. J Cell Sci. 1993 Feb;104(Pt 2):327–337. doi: 10.1242/jcs.104.2.327. [DOI] [PubMed] [Google Scholar]
  42. Schnapp A., Clos J., Hädelt W., Schreck R., Cvekl A., Grummt I. Isolation and functional characterization of TIF-IB, a factor that confers promoter specificity to mouse RNA polymerase I. Nucleic Acids Res. 1990 Mar 25;18(6):1385–1393. doi: 10.1093/nar/18.6.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Schwartz L. B., Roeder R. G. Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase I from the mouse myeloma, MOPC 315. J Biol Chem. 1974 Sep 25;249(18):5898–5906. [PubMed] [Google Scholar]
  46. 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]
  47. Sugimoto A., Iino Y., Maeda T., Watanabe Y., Yamamoto M. Schizosaccharomyces pombe ste11+ encodes a transcription factor with an HMG motif that is a critical regulator of sexual development. Genes Dev. 1991 Nov;5(11):1990–1999. doi: 10.1101/gad.5.11.1990. [DOI] [PubMed] [Google Scholar]
  48. Tower J., Culotta V. C., Sollner-Webb B. Factors and nucleotide sequences that direct ribosomal DNA transcription and their relationship to the stable transcription complex. Mol Cell Biol. 1986 Oct;6(10):3451–3462. doi: 10.1128/mcb.6.10.3451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Voit R., Schnapp A., Kuhn A., Rosenbauer H., Hirschmann P., Stunnenberg H. G., Grummt I. The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation. EMBO J. 1992 Jun;11(6):2211–2218. doi: 10.1002/j.1460-2075.1992.tb05280.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Walker J. M., Gooderham K., Hastings J. R., Mayes E., Johns E. W. The primary structures of non-histone chromosomal proteins HMG 1 and 2. FEBS Lett. 1980 Dec 29;122(2):264–270. doi: 10.1016/0014-5793(80)80453-4. [DOI] [PubMed] [Google Scholar]
  52. Waterman M. L., Fischer W. H., Jones K. A. A thymus-specific member of the HMG protein family regulates the human T cell receptor C alpha enhancer. Genes Dev. 1991 Apr;5(4):656–669. doi: 10.1101/gad.5.4.656. [DOI] [PubMed] [Google Scholar]
  53. van de Wetering M., Oosterwegel M., Dooijes D., Clevers H. Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J. 1991 Jan;10(1):123–132. doi: 10.1002/j.1460-2075.1991.tb07928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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