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
. 1977 Jan;74(1):44–48. doi: 10.1073/pnas.74.1.44

Selective and accurate transcription of the Xenopus laevis 5S RNA genes in isolated chromatin by purified RNA polymerase III.

C S Parker, R G Roeder
PMCID: PMC393193  PMID: 264693

Abstract

Chromatin isolated from immature oocytes was found to contain an endogenous RNA polymerase activity (RNA nucleotidyltransferase; nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) that synthesizes predominately 5S RNA. However, the levels of total RNA synthesis and 5S RNA synthesis in chromatin were each stimulated 10- to 50-fold by an exogenous RNA polymerase III purified from X. laevis oocytes. The 5S genes in chromatin were transcribed by the exogenous enzyme in a highly selective (3000-fold above random) and predominately asymmetric fashion. A significant fraction of 5S RNA sequences were also found in a discrete transcript, approximately 5S in size. Total RNA synthesis was significantly stimulated when chromatin was transcribed by oocyte RNA polymerase I, murine RNA polymerase II, and low levels of Escherichia coli RNA polymerase. However, these enzymes did not significantly stimulate 5S RNA synthesis above the endogenous levels. Both homologous oocyte RNA polymerase I and III and E. coli RNA polymerase transcribed the 5S genes in deproteinized DNA to approximately the same extent (severalfold above random) and both the sense and anti-sense strands of the gene were transcribed. It appears, therefore, that both chromatin-associated components and a purified RNA polymerase III are necessary and sufficient for the selective and accurate transcription of the 5S RNA genes in vitro.

Full text

PDF
44

Images in this article

46Image
on p.46

Selected References

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

  1. Axel R., Cedar H., Felsenfeld G. Synthesis of globin ribonucleic acid from duck-reticulocyte chromatin in vitro. Proc Natl Acad Sci U S A. 1973 Jul;70(7):2029–2032. doi: 10.1073/pnas.70.7.2029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown D. D., Sugimoto K. 5 S DNAs of Xenopus laevis and Xenopus mulleri: evolution of a gene family. J Mol Biol. 1973 Aug 15;78(3):397–415. doi: 10.1016/0022-2836(73)90464-6. [DOI] [PubMed] [Google Scholar]
  3. Brownlee G. G., Cartwright E. M., Brown D. D. Sequence studies of the 5 S DNA of Xenopus laevis. J Mol Biol. 1974 Nov 15;89(4):703–718. doi: 10.1016/0022-2836(74)90046-1. [DOI] [PubMed] [Google Scholar]
  4. Carroll D., Brown D. D. Adjacent repeating units of Xenopus laevis 5S DNA can be heterogeneous in length. Cell. 1976 Apr;7(4):477–486. doi: 10.1016/0092-8674(76)90199-9. [DOI] [PubMed] [Google Scholar]
  5. Denis H., Wegnez M. Recherches biochimiques sur l'oogenése. 7. Synthése et maturation du RNA 5S dans les petitis oocytes de Xenopus laevis. Biochimie. 1973;55(9):1137–1151. doi: 10.1016/s0300-9084(73)80453-5. [DOI] [PubMed] [Google Scholar]
  6. Dumont J. N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol. 1972 Feb;136(2):153–179. doi: 10.1002/jmor.1051360203. [DOI] [PubMed] [Google Scholar]
  7. Ford P. J., Southern E. M. Different sequences for 5S RNA in kidney cells and ovaries of Xenopus laevis. Nat New Biol. 1973 Jan 3;241(105):7–12. doi: 10.1038/newbio241007a0. [DOI] [PubMed] [Google Scholar]
  8. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  9. Gilmour R. S., Paul J. Tissue-specific transcription of the globin gene in isolated chromatin. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3440–3442. doi: 10.1073/pnas.70.12.3440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Honjo T., Reeder R. H. Transcription of Xenopus chromatin by homologous ribonucleic acid polymerase: aberrant synthesis of ribosomal and 5S ribonucleic acid. Biochemistry. 1974 Apr 23;13(9):1896–1899. doi: 10.1021/bi00706a018. [DOI] [PubMed] [Google Scholar]
  11. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  12. Reeder R. H., Roeder R. G. Ribosomal RNA synthesis in isolated nuclei. J Mol Biol. 1972 Jun 28;67(3):433–441. doi: 10.1016/0022-2836(72)90461-5. [DOI] [PubMed] [Google Scholar]
  13. Reeder R. H. Transcription of chromatin by bacterial RNA polymerase. J Mol Biol. 1973 Oct 25;80(2):229–241. doi: 10.1016/0022-2836(73)90169-1. [DOI] [PubMed] [Google Scholar]
  14. Robertson H. D., Hunter T. Sensitive methods for the detection and characterization of double helical ribonucleic acid. J Biol Chem. 1975 Jan 25;250(2):418–425. [PubMed] [Google Scholar]
  15. Roeder R. G. Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus laevis. Levels of activity during oocyte and embryonic development. J Biol Chem. 1974 Jan 10;249(1):249–256. [PubMed] [Google Scholar]
  16. Sklar V. E., Roeder R. G. Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase III from the mouse plasmacytoma, MOPC 315. J Biol Chem. 1976 Feb 25;251(4):1064–1073. [PubMed] [Google Scholar]
  17. Sklar V. E., Schwartz L. B., Roeder R. G. Distinct molecular structures of nuclear class I, II, and III DNA-dependent RNA polymerases. Proc Natl Acad Sci U S A. 1975 Jan;72(1):348–352. doi: 10.1073/pnas.72.1.348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stein G., Park W., Thrall C., Mans R., Stein J. Regulation of cell cycle stage-specific transcription of histone genes from chromatin by non-histone chromosomal proteins. Nature. 1975 Oct 30;257(5529):764–767. doi: 10.1038/257764a0. [DOI] [PubMed] [Google Scholar]
  19. Thomas C. RNA metabolism in previtellogenic oocytes of Xenopus laevis. Dev Biol. 1974 Aug;39(2):191–197. doi: 10.1016/0012-1606(74)90234-6. [DOI] [PubMed] [Google Scholar]
  20. Timmis K., Cabello F., Cohen S. N. Utilization of two distinct modes of replication by a hybrid plasmid constructed in vitro from separate replicons. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4556–4560. doi: 10.1073/pnas.71.11.4556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tsai M. J., Towle T. C., Harris S. E., O'Malley B. W. Effect of estrogen on gene expression in the chick oviduct. J Biol Chem. 1976 Apr 10;251(7):1960–1968. [PubMed] [Google Scholar]
  22. Weil P. A., Blatti S. P. HeLa cell deoxyribonucleic acid dependent RNA polymerases: function and properties of the class III enzymes. Biochemistry. 1976 Apr 6;15(7):1500–1509. doi: 10.1021/bi00652a022. [DOI] [PubMed] [Google Scholar]
  23. Weinmann R., Brendler T. G., Raskas H. J., Roeder R. G. Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection. Cell. 1976 Apr;7(4):557–566. doi: 10.1016/0092-8674(76)90206-3. [DOI] [PubMed] [Google Scholar]
  24. Weinmann R., Roeder R. G. Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes. Proc Natl Acad Sci U S A. 1974 May;71(5):1790–1794. doi: 10.1073/pnas.71.5.1790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  26. Wilson G. N., Steggles A. W., Nienhuis A. W. Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4835–4839. doi: 10.1073/pnas.72.12.4835. [DOI] [PMC free article] [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