Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Nov;81(21):6672–6675. doi: 10.1073/pnas.81.21.6672

A purified fraction containing RNA polymerase I that can accurately transcribe rat ribosomal RNA gene.

R N Kurl, L I Rothblum, S T Jacob
PMCID: PMC391992  PMID: 6593724

Abstract

Transcription of a cloned rat rDNA containing the transcription initiation region was studied using purified fractions derived from whole cell extract. The cell extract obtained from rat mammary adenocarcinoma cells was fractionated successively on DEAE-Sephadex and heparin-Sepharose columns. The fractions eluted by 175 mM (NH4)2SO4 (DE-B) from the DEAE-Sephadex column and those eluting with RNA polymerase I on the subsequent heparin-Sepharose column (HS-B) were used in a run-off transcription assay using Xho I-cleaved cloned rat rDNA. Both fractions that contained greater than 90% of the RNA polymerase I activity produced the anticipated 635-nucleotide-long transcript. Fractionation of fraction DE-B on a phosphocellulose column also resulted in a RNA polymerase I-containing complex that, by itself, could transcribe the rDNA accurately. The major polypeptides obtained after NaDodSO4/PAGE of fraction HS-B had Mr values of 190,000, 120,000, 65,000, 42,000, and 25,000, corresponding to the major subunits of RNA polymerase I. Purified nuclear RNA polymerase I did not produce the correct transcript. These studies have shown that a purified fraction containing RNA polymerase I from whole cells can support accurate transcription of rDNA.

Full text

PDF
6672

Images in this article

6673Image
on p.6673
6674Image
on p.6674

Selected References

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

  1. Duceman B. W., Rose K. M., Jacob S. T. Activation of purified hepatoma RNA polymerase I by homologous protein kinase NII. J Biol Chem. 1981 Nov 10;256(21):10755–10758. [PubMed] [Google Scholar]
  2. Dynan W. S., Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell. 1983 Mar;32(3):669–680. doi: 10.1016/0092-8674(83)90053-3. [DOI] [PubMed] [Google Scholar]
  3. Engelke D. R., Ng S. Y., Shastry B. S., Roeder R. G. Specific interaction of a purified transcription factor with an internal control region of 5S RNA genes. Cell. 1980 Mar;19(3):717–728. doi: 10.1016/s0092-8674(80)80048-1. [DOI] [PubMed] [Google Scholar]
  4. Grummt I. Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vivo. Proc Natl Acad Sci U S A. 1981 Feb;78(2):727–731. doi: 10.1073/pnas.78.2.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kohorn B. D., Rae P. M. Accurate transcription of truncated ribosomal DNA templates in a Drosophila cell-free system. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1501–1505. doi: 10.1073/pnas.79.5.1501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  7. Learned R. M., Tjian R. In vitro transcription of human ribosomal RNA genes by RNA polymerase I. J Mol Appl Genet. 1982;1(6):575–584. [PubMed] [Google Scholar]
  8. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Miller K. G., Sollner-Webb B. Transcription of mouse rRNA genes by RNA polymerase I: in vitro and in vivo initiation and processing sites. Cell. 1981 Nov;27(1 Pt 2):165–174. doi: 10.1016/0092-8674(81)90370-6. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Rose K. M., Ruch P. A., Morris H. P., Jacob S. T. RNA polymerases from a rat hepatoma. Partial purification and comparison of properties with corresponding liver enzymes. Biochim Biophys Acta. 1976 Apr 15;432(1):60–72. doi: 10.1016/0005-2787(76)90041-1. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Rothblum L. I., Reddy R., Cassidy B. Transcription initiation site of rat ribosomal DNA. Nucleic Acids Res. 1982 Nov 25;10(22):7345–7362. doi: 10.1093/nar/10.22.7345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sakonju S., Bogenhagen D. F., Brown D. D. A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5' border of the region. Cell. 1980 Jan;19(1):13–25. doi: 10.1016/0092-8674(80)90384-0. [DOI] [PubMed] [Google Scholar]
  16. Samuels M., Fire A., Sharp P. A. Separation and characterization of factors mediating accurate transcription by RNA polymerase II. J Biol Chem. 1982 Dec 10;257(23):14419–14427. [PubMed] [Google Scholar]
  17. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  18. Segall J., Matsui T., Roeder R. G. Multiple factors are required for the accurate transcription of purified genes by RNA polymerase III. J Biol Chem. 1980 Dec 25;255(24):11986–11991. [PubMed] [Google Scholar]
  19. Shastry B. S., Ng S. Y., Roeder R. G. Multiple factors involved in the transcription of class III genes in Xenopus laevis. J Biol Chem. 1982 Nov 10;257(21):12979–12986. [PubMed] [Google Scholar]
  20. Sollner-Webb B., Reeder R. H. The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis. Cell. 1979 Oct;18(2):485–499. doi: 10.1016/0092-8674(79)90066-7. [DOI] [PubMed] [Google Scholar]
  21. Swanson M. E., Holland M. J. RNA polymerase I-dependent selective transcription of yeast ribosomal DNA. Identification of a new cellular ribosomal RNA precursor. J Biol Chem. 1983 Mar 10;258(5):3242–3250. [PubMed] [Google Scholar]
  22. Tsai S. Y., Tsai M. J., Kops L. E., Minghetti P. P., O'Malley B. W. Transcription factors from oviduct and HeLa cells are similar. J Biol Chem. 1981 Dec 25;256(24):13055–13059. [PubMed] [Google Scholar]
  23. Weil P. A., Luse D. S., Segall J., Roeder R. G. Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell. 1979 Oct;18(2):469–484. doi: 10.1016/0092-8674(79)90065-5. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES