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. 1989 May;9(5):2018–2024. doi: 10.1128/mcb.9.5.2018

Identification of a 150-kilodalton polypeptide that copurifies with yeast TFIIIC and binds specifically to tRNA genes.

D L Johnson 1, S L Wilson 1
PMCID: PMC362994  PMID: 2664466

Abstract

The transcription in vitro of eucaryotic tRNA genes by RNA polymerase III requires two transcription factors, designated TFIIIB and TFIIIC. One of the critical functions of TFIIIC in the transcription of tRNA genes is that it interacts directly and specifically with the two internal promoter elements of these genes. We have partially purified Saccharomyces cerevisiae TFIIIC by chromatography on Bio-Rex 70, DEAE-cellulose, and phosphocellulose resins. A 150-kilodalton (kDa) DNA-binding polypeptide copurified with TFIIIC activity. This 150-kDa protein coeluted with the DNA-binding activity of TFIIIC after rechromatography of TFIIIC on phosphocellulose and its elution with a linear salt gradient. The stable and high-affinity interaction of this protein with tRNA genes was demonstrated by the maintenance of a protein-DNA complex under conditions of high ionic strength. Finally, we showed by two criteria that the interaction of this protein with tRNA genes was specific. First, the protein-DNA complex was competed with only by DNA-containing tRNA genes; second, the protein preferentially bound to DNA fragments containing a tRNA gene. These results strongly suggest that the DNA-binding domain of the yeast TFIIIC is contained within this 150-kDa polypeptide.

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Selected References

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

  1. Baker R. E., Gabrielsen O., Hall B. D. Effects of tRNATyr point mutations on the binding of yeast RNA polymerase III transcription factor C. J Biol Chem. 1986 Apr 25;261(12):5275–5282. [PubMed] [Google Scholar]
  2. Boulanger P. A., Yoshinaga S. K., Berk A. J. DNA-binding properties and characterization of human transcription factor TFIIIC2. J Biol Chem. 1987 Nov 5;262(31):15098–15105. [PubMed] [Google Scholar]
  3. Burke D. J., Söll D. Functional analysis of fractionated Drosophila Kc cell tRNA gene transcription components. J Biol Chem. 1985 Jan 25;260(2):816–823. [PubMed] [Google Scholar]
  4. Camier S., Gabrielsen O., Baker R., Sentenac A. A split binding site for transcription factor tau on the tRNA3Glu gene. EMBO J. 1985 Feb;4(2):491–500. doi: 10.1002/j.1460-2075.1985.tb03655.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlson D. P., Ross J. Point mutation associated with hereditary persistence of fetal hemoglobin decreases RNA polymerase III transcription upstream of the affected gamma-globin gene. Mol Cell Biol. 1986 Sep;6(9):3278–3282. doi: 10.1128/mcb.6.9.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung J., Sussman D. J., Zeller R., Leder P. The c-myc gene encodes superimposed RNA polymerase II and III promoters. Cell. 1987 Dec 24;51(6):1001–1008. doi: 10.1016/0092-8674(87)90586-1. [DOI] [PubMed] [Google Scholar]
  7. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jahn D., Wingender E., Seifart K. H. Transcription complexes for various class III genes differ in parameters of formation and stability towards salt. J Mol Biol. 1987 Jan 20;193(2):303–313. doi: 10.1016/0022-2836(87)90221-x. [DOI] [PubMed] [Google Scholar]
  9. Klekamp M. S., Weil P. A. Specific transcription of homologous class III genes in yeast-soluble cell-free extracts. J Biol Chem. 1982 Jul 25;257(14):8432–8441. [PubMed] [Google Scholar]
  10. Klemenz R., Stillman D. J., Geiduschek E. P. Specific interactions of Saccharomyces cerevisiae proteins with a promoter region of eukaryotic tRNA genes. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6191–6195. doi: 10.1073/pnas.79.20.6191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Larson D., Bradford-Wilcox J., Young L. S., Sprague K. U. A short 5' flanking region containing conserved sequences is required for silkworm alanine tRNA gene activity. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3416–3420. doi: 10.1073/pnas.80.11.3416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lassar A. B., Martin P. L., Roeder R. G. Transcription of class III genes: formation of preinitiation complexes. Science. 1983 Nov 18;222(4625):740–748. doi: 10.1126/science.6356356. [DOI] [PubMed] [Google Scholar]
  14. Marzouki N., Camier S., Ruet A., Moenne A., Sentenac A. Selective proteolysis defines two DNA binding domains in yeast transcription factor tau. Nature. 1986 Sep 11;323(6084):176–178. doi: 10.1038/323176a0. [DOI] [PubMed] [Google Scholar]
  15. Miskimins W. K., Roberts M. P., McClelland A., Ruddle F. H. Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6741–6744. doi: 10.1073/pnas.82.20.6741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ottonello S., Rivier D. H., Doolittle G. M., Young L. S., Sprague K. U. The properties of a new polymerase III transcription factor reveal that transcription complexes can assemble by more than one pathway. EMBO J. 1987 Jul;6(7):1921–1927. doi: 10.1002/j.1460-2075.1987.tb02452.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Raymond K. C., Raymond G. J., Johnson J. D. In vivo modulation of yeast tRNA gene expression by 5'-flanking sequences. EMBO J. 1985 Oct;4(10):2649–2656. doi: 10.1002/j.1460-2075.1985.tb03983.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ruet A., Camier S., Smagowicz W., Sentenac A., Fromageot P. Isolation of a class C transcription factor which forms a stable complex with tRNA genes. EMBO J. 1984 Feb;3(2):343–350. doi: 10.1002/j.1460-2075.1984.tb01809.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schaack J., Sharp S., Dingermann T., Burke D. J., Cooley L., Söll D. The extent of a eukaryotic tRNA gene. 5'- and 3'-flanking sequence dependence for transcription and stable complex formation. J Biol Chem. 1984 Feb 10;259(3):1461–1467. [PubMed] [Google Scholar]
  20. Schaack J., Sharp S., Dingermann T., Söll D. Transcription of eukaryotic tRNA genes in vitro. II. Formation of stable complexes. J Biol Chem. 1983 Feb 25;258(4):2447–2453. [PubMed] [Google Scholar]
  21. 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]
  22. Sharp S. J., Schaack J., Cooley L., Burke D. J., Söll D. Structure and transcription of eukaryotic tRNA genes. CRC Crit Rev Biochem. 1985;19(2):107–144. doi: 10.3109/10409238509082541. [DOI] [PubMed] [Google Scholar]
  23. Stillman D. J., Better M., Geiduschek E. P. Electron-microscopic examination of the binding of a large RNA polymerase III transcription factor to a tRNA gene. J Mol Biol. 1985 Sep 20;185(2):451–455. doi: 10.1016/0022-2836(85)90417-6. [DOI] [PubMed] [Google Scholar]
  24. Taylor M. J., Segall J. Characterization of factors and DNA sequences required for accurate transcription of the Saccharomyces cerevisiae 5 S RNA gene. J Biol Chem. 1985 Apr 10;260(7):4531–4540. [PubMed] [Google Scholar]
  25. Thomas P. S., Farquhar M. N. Specific measurement of DNA in nuclei and nucleic acids using diaminobenzoic acid. Anal Biochem. 1978 Aug 15;89(1):35–44. doi: 10.1016/0003-2697(78)90724-8. [DOI] [PubMed] [Google Scholar]
  26. Watt R. A., Shatzman A. R., Rosenberg M. Expression and characterization of the human c-myc DNA-binding protein. Mol Cell Biol. 1985 Mar;5(3):448–456. doi: 10.1128/mcb.5.3.448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Willis I., Hottinger H., Pearson D., Chisholm V., Leupold U., Söll D. Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor. EMBO J. 1984 Jul;3(7):1573–1580. doi: 10.1002/j.1460-2075.1984.tb02013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yoshinaga S. K., Boulanger P. A., Berk A. J. Resolution of human transcription factor TFIIIC into two functional components. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3585–3589. doi: 10.1073/pnas.84.11.3585. [DOI] [PMC free article] [PubMed] [Google Scholar]

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