Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Jul 1;17(13):3692–3703. doi: 10.1093/emboj/17.13.3692

A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription.

P Milkereit 1, H Tschochner 1
PMCID: PMC1170705  PMID: 9649439

Abstract

Only a small proportion (<2%) of RNA polymerase I (pol I) from whole-cell extracts appeared to be competent for specific initiation at the ribosomal gene promoter in a yeast reconstituted transcription system. Initiation-competent pol I molecules were found exclusively in salt-resistant complexes that contain the pol I-specific initiation factor Rrn3p. Levels of initiation-competent complexes in extracts were independent of total Rrn3p content and varied with the growth state of the cells. Although extracts from stationary phase cells contained substantial amounts of Rrn3p and pol I, they lacked the pol I-Rrn3p complex and were inactive in promoter-dependent transcription. Activity was restored by adding purified pol I-Rrn3p complex to extracts from stationary phase cells. The pol I-Rrn3p complex dissociated during transcription and lost its capacity for subsequent reinitiation in vitro, suggesting a stoichiometric rather than a catalytic activity in initiation. We propose that the formation and disruption of the pol I-Rrn3p complex reflects a molecular switch for regulating rRNA synthesis and its growth rate-dependent regulation.

Full Text

The Full Text of this article is available as a PDF (408.1 KB).

Selected References

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

  1. Akhtar A., Faye G., Bentley D. L. Distinct activated and non-activated RNA polymerase II complexes in yeast. EMBO J. 1996 Sep 2;15(17):4654–4664. [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Beckmann H., Chen J. L., O'Brien T., Tjian R. Coactivator and promoter-selective properties of RNA polymerase I TAFs. Science. 1995 Dec 1;270(5241):1506–1509. doi: 10.1126/science.270.5241.1506. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Buttgereit D., Pflugfelder G., Grummt I. Growth-dependent regulation of rRNA synthesis is mediated by a transcription initiation factor (TIF-IA). Nucleic Acids Res. 1985 Nov 25;13(22):8165–8180. doi: 10.1093/nar/13.22.8165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cavanaugh A. H., Hempel W. M., Taylor L. J., Rogalsky V., Todorov G., Rothblum L. I. Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product. Nature. 1995 Mar 9;374(6518):177–180. doi: 10.1038/374177a0. [DOI] [PubMed] [Google Scholar]
  7. Cavanaugh A. H., Thompson E. A., Jr Hormonal regulation of transcription of rDNA: glucocorticoid effects upon initiation and elongation in vitro. Nucleic Acids Res. 1985 May 10;13(9):3357–3369. doi: 10.1093/nar/13.9.3357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chasman D. I., Leatherwood J., Carey M., Ptashne M., Kornberg R. D. Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro. Mol Cell Biol. 1989 Nov;9(11):4746–4749. doi: 10.1128/mcb.9.11.4746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Gokal P. K., Mahajan P. B., Thompson E. A. Hormonal regulation of transcription of rDNA. Formation of initiated complexes by RNA polymerase I in vitro. J Biol Chem. 1990 Sep 25;265(27):16234–16243. [PubMed] [Google Scholar]
  14. Grandi P., Doye V., Hurt E. C. Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96. EMBO J. 1993 Aug;12(8):3061–3071. doi: 10.1002/j.1460-2075.1993.tb05975.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Keener J., Dodd J. A., Lalo D., Nomura M. Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13458–13462. doi: 10.1073/pnas.94.25.13458. [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. 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]
  20. Lobo S. M., Tanaka M., Sullivan M. L., Hernandez N. A TBP complex essential for transcription from TATA-less but not TATA-containing RNA polymerase III promoters is part of the TFIIIB fraction. Cell. 1992 Dec 11;71(6):1029–1040. doi: 10.1016/0092-8674(92)90397-u. [DOI] [PubMed] [Google Scholar]
  21. Mahajan P. B., Gokal P. K., Thompson E. A. Hormonal regulation of transcription of rDNA. The role of TFIC in formation of initiation complexes. J Biol Chem. 1990 Sep 25;265(27):16244–16247. [PubMed] [Google Scholar]
  22. Mahajan P. B., Thompson E. A. Hormonal regulation of transcription of rDNA. Purification and characterization of the hormone-regulated transcription factor IC. J Biol Chem. 1990 Sep 25;265(27):16225–16233. [PubMed] [Google Scholar]
  23. Milkereit P., Schultz P., Tschochner H. Resolution of RNA polymerase I into dimers and monomers and their function in transcription. Biol Chem. 1997 Dec;378(12):1433–1443. doi: 10.1515/bchm.1997.378.12.1433. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. O'Mahony D. J., Xie W. Q., Smith S. D., Singer H. A., Rothblum L. I. Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation. In vitro dephosphorylation of UBF reduces its transactivation properties. J Biol Chem. 1992 Jan 5;267(1):35–38. [PubMed] [Google Scholar]
  26. Paule M. R. Regulation of ribosomal RNA transcription during differentiation of Acanthamoeba castellanii: a review. J Protozool. 1983 May;30(2):211–214. doi: 10.1111/j.1550-7408.1983.tb02905.x. [DOI] [PubMed] [Google Scholar]
  27. Posnett D. N., Tam J. P. Multiple antigenic peptide method for producing antipeptide site-specific antibodies. Methods Enzymol. 1989;178:739–746. doi: 10.1016/0076-6879(89)78048-4. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Roeder R. G. Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus laevis. Isolation and partial characterization. J Biol Chem. 1974 Jan 10;249(1):241–248. [PubMed] [Google Scholar]
  31. Saez-Vasquez J., Pikaard C. S. Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11869–11874. doi: 10.1073/pnas.94.22.11869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sayre M. H., Tschochner H., Kornberg R. D. Purification and properties of Saccharomyces cerevisiae RNA polymerase II general initiation factor a. J Biol Chem. 1992 Nov 15;267(32):23383–23387. [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Schnapp A., Pfleiderer C., Rosenbauer H., Grummt I. A growth-dependent transcription initiation factor (TIF-IA) interacting with RNA polymerase I regulates mouse ribosomal RNA synthesis. EMBO J. 1990 Sep;9(9):2857–2863. doi: 10.1002/j.1460-2075.1990.tb07475.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schnapp A., Schnapp G., Erny B., Grummt I. Function of the growth-regulated transcription initiation factor TIF-IA in initiation complex formation at the murine ribosomal gene promoter. Mol Cell Biol. 1993 Nov;13(11):6723–6732. doi: 10.1128/mcb.13.11.6723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Seither P., Iben S., Grummt I. Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors. J Mol Biol. 1998 Jan 9;275(1):43–53. doi: 10.1006/jmbi.1997.1434. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Stenbeck G., Harter C., Brecht A., Herrmann D., Lottspeich F., Orci L., Wieland F. T. beta'-COP, a novel subunit of coatomer. EMBO J. 1993 Jul;12(7):2841–2845. doi: 10.1002/j.1460-2075.1993.tb05945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stevens A. R., Pachler P. F. RNA synthesis and turnover during density-inhibited growth and encystment of Acanthamoeba castellanii. J Cell Biol. 1973 May;57(2):525–537. doi: 10.1083/jcb.57.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stewart S. E., Roeder G. S. Transcription by RNA polymerase I stimulates mitotic recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Aug;9(8):3464–3472. doi: 10.1128/mcb.9.8.3464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taggart A. K., Fisher T. S., Pugh B. F. The TATA-binding protein and associated factors are components of pol III transcription factor TFIIIB. Cell. 1992 Dec 11;71(6):1015–1028. doi: 10.1016/0092-8674(92)90396-t. [DOI] [PubMed] [Google Scholar]
  44. Tanese N., Pugh B. F., Tjian R. Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex. Genes Dev. 1991 Dec;5(12A):2212–2224. doi: 10.1101/gad.5.12a.2212. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Tschochne H., Milkereit P. RNA polymerase I from S. cerevisiae depends on an additional factor to release terminated transcripts from the template. FEBS Lett. 1997 Jun 30;410(2-3):461–466. doi: 10.1016/s0014-5793(97)00636-4. [DOI] [PubMed] [Google Scholar]
  47. Tschochner H. A novel RNA polymerase I-dependent RNase activity that shortens nascent transcripts from the 3' end. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12914–12919. doi: 10.1073/pnas.93.23.12914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Voit R., Schäfer K., Grummt I. Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein. Mol Cell Biol. 1997 Aug;17(8):4230–4237. doi: 10.1128/mcb.17.8.4230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Werner-Washburne M., Braun E., Johnston G. C., Singer R. A. Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol Rev. 1993 Jun;57(2):383–401. doi: 10.1128/mr.57.2.383-401.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Zhai W., Tuan J. A., Comai L. SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription. Genes Dev. 1997 Jun 15;11(12):1605–1617. doi: 10.1101/gad.11.12.1605. [DOI] [PubMed] [Google Scholar]
  53. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES