Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Mar;15(3):1234–1243. doi: 10.1128/mcb.15.3.1234

Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription.

J J Kang 1, D T Auble 1, J A Ranish 1, S Hahn 1
PMCID: PMC230346  PMID: 7862117

Abstract

To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.

Full Text

The Full Text of this article is available as a PDF (585.7 KB).

Selected References

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

  1. Auble D. T., Hahn S. An ATP-dependent inhibitor of TBP binding to DNA. Genes Dev. 1993 May;7(5):844–856. doi: 10.1101/gad.7.5.844. [DOI] [PubMed] [Google Scholar]
  2. Auble D. T., Hansen K. E., Mueller C. G., Lane W. S., Thorner J., Hahn S. Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 1994 Aug 15;8(16):1920–1934. doi: 10.1101/gad.8.16.1920. [DOI] [PubMed] [Google Scholar]
  3. Bass S. H., Mulkerrin M. G., Wells J. A. A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4498–4502. doi: 10.1073/pnas.88.10.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett W. F., Paoni N. F., Keyt B. A., Botstein D., Jones A. J., Presta L., Wurm F. M., Zoller M. J. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem. 1991 Mar 15;266(8):5191–5201. [PubMed] [Google Scholar]
  5. Brow D. A., Guthrie C. Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes Dev. 1990 Aug;4(8):1345–1356. doi: 10.1101/gad.4.8.1345. [DOI] [PubMed] [Google Scholar]
  6. Buratowski S., Hahn S., Guarente L., Sharp P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. doi: 10.1016/0092-8674(89)90578-3. [DOI] [PubMed] [Google Scholar]
  7. Buratowski S., Zhou H. Transcription factor IID mutants defective for interaction with transcription factor IIA. Science. 1992 Feb 28;255(5048):1130–1132. doi: 10.1126/science.1546314. [DOI] [PubMed] [Google Scholar]
  8. Chen W., Tabor S., Struhl K. Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase. Cell. 1987 Sep 25;50(7):1047–1055. doi: 10.1016/0092-8674(87)90171-1. [DOI] [PubMed] [Google Scholar]
  9. Chi T., Carey M. The ZEBRA activation domain: modular organization and mechanism of action. Mol Cell Biol. 1993 Nov;13(11):7045–7055. doi: 10.1128/mcb.13.11.7045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chodosh L. A., Carthew R. W., Sharp P. A. A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor. Mol Cell Biol. 1986 Dec;6(12):4723–4733. doi: 10.1128/mcb.6.12.4723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Conaway J. W., Conaway R. C. A multisubunit transcription factor essential for accurate initiation by RNA polymerase II. J Biol Chem. 1989 Feb 5;264(4):2357–2362. [PubMed] [Google Scholar]
  12. Cormack B. P., Strubin M., Stargell L. A., Struhl K. Conserved and nonconserved functions of the yeast and human TATA-binding proteins. Genes Dev. 1994 Jun 1;8(11):1335–1343. doi: 10.1101/gad.8.11.1335. [DOI] [PubMed] [Google Scholar]
  13. Cormack B. P., Struhl K. The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells. Cell. 1992 May 15;69(4):685–696. doi: 10.1016/0092-8674(92)90232-2. [DOI] [PubMed] [Google Scholar]
  14. Cortes P., Flores O., Reinberg D. Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ. Mol Cell Biol. 1992 Jan;12(1):413–421. doi: 10.1128/mcb.12.1.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  16. DeJong J., Roeder R. G. A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA. Genes Dev. 1993 Nov;7(11):2220–2234. doi: 10.1101/gad.7.11.2220. [DOI] [PubMed] [Google Scholar]
  17. Gibbs C. S., Zoller M. J. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J Biol Chem. 1991 May 15;266(14):8923–8931. [PubMed] [Google Scholar]
  18. Hahn S., Buratowski S., Sharp P. A., Guarente L. Identification of a yeast protein homologous in function to the mammalian general transcription factor, TFIIA. EMBO J. 1989 Nov;8(11):3379–3382. doi: 10.1002/j.1460-2075.1989.tb08501.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hahn S., Buratowski S., Sharp P. A., Guarente L. Isolation of the gene encoding the yeast TATA binding protein TFIID: a gene identical to the SPT15 suppressor of Ty element insertions. Cell. 1989 Sep 22;58(6):1173–1181. doi: 10.1016/0092-8674(89)90515-1. [DOI] [PubMed] [Google Scholar]
  20. Hahn S., Hoar E. T., Guarente L. Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8562–8566. doi: 10.1073/pnas.82.24.8562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hope I. A., Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986 Sep 12;46(6):885–894. doi: 10.1016/0092-8674(86)90070-x. [DOI] [PubMed] [Google Scholar]
  22. Inostroza J. A., Mermelstein F. H., Ha I., Lane W. S., Reinberg D. Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription. Cell. 1992 Aug 7;70(3):477–489. doi: 10.1016/0092-8674(92)90172-9. [DOI] [PubMed] [Google Scholar]
  23. Kim J. L., Nikolov D. B., Burley S. K. Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature. 1993 Oct 7;365(6446):520–527. doi: 10.1038/365520a0. [DOI] [PubMed] [Google Scholar]
  24. Kim Y., Geiger J. H., Hahn S., Sigler P. B. Crystal structure of a yeast TBP/TATA-box complex. Nature. 1993 Oct 7;365(6446):512–520. doi: 10.1038/365512a0. [DOI] [PubMed] [Google Scholar]
  25. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  26. Lieberman P. M., Berk A. J. A mechanism for TAFs in transcriptional activation: activation domain enhancement of TFIID-TFIIA--promoter DNA complex formation. Genes Dev. 1994 May 1;8(9):995–1006. doi: 10.1101/gad.8.9.995. [DOI] [PubMed] [Google Scholar]
  27. Ma D., Watanabe H., Mermelstein F., Admon A., Oguri K., Sun X., Wada T., Imai T., Shiroya T., Reinberg D. Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription. Genes Dev. 1993 Nov;7(11):2246–2257. doi: 10.1101/gad.7.11.2246. [DOI] [PubMed] [Google Scholar]
  28. Maldonado E., Ha I., Cortes P., Weis L., Reinberg D. Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex. Mol Cell Biol. 1990 Dec;10(12):6335–6347. doi: 10.1128/mcb.10.12.6335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Meissner W., Holland R., Waldschmidt R., Seifart K. H. Transcription factor IIA stimulates the expression of classical polIII-genes. Nucleic Acids Res. 1993 Feb 25;21(4):1013–1018. doi: 10.1093/nar/21.4.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meisterernst M., Roeder R. G. Family of proteins that interact with TFIID and regulate promoter activity. Cell. 1991 Nov 1;67(3):557–567. doi: 10.1016/0092-8674(91)90530-c. [DOI] [PubMed] [Google Scholar]
  32. Meisterernst M., Roy A. L., Lieu H. M., Roeder R. G. Activation of class II gene transcription by regulatory factors is potentiated by a novel activity. Cell. 1991 Sep 6;66(5):981–993. doi: 10.1016/0092-8674(91)90443-3. [DOI] [PubMed] [Google Scholar]
  33. Merino A., Madden K. R., Lane W. S., Champoux J. J., Reinberg D. DNA topoisomerase I is involved in both repression and activation of transcription. Nature. 1993 Sep 16;365(6443):227–232. doi: 10.1038/365227a0. [DOI] [PubMed] [Google Scholar]
  34. Ranish J. A., Hahn S. The yeast general transcription factor TFIIA is composed of two polypeptide subunits. J Biol Chem. 1991 Oct 15;266(29):19320–19327. [PubMed] [Google Scholar]
  35. Ranish J. A., Lane W. S., Hahn S. Isolation of two genes that encode subunits of the yeast transcription factor IIA. Science. 1992 Feb 28;255(5048):1127–1129. doi: 10.1126/science.1546313. [DOI] [PubMed] [Google Scholar]
  36. Reinberg D., Horikoshi M., Roeder R. G. Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site. J Biol Chem. 1987 Mar 5;262(7):3322–3330. [PubMed] [Google Scholar]
  37. Sawadogo M., Roeder R. G. Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4394–4398. doi: 10.1073/pnas.82.13.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Schuler G. D., Altschul S. F., Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. doi: 10.1002/prot.340090304. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Shen S. H., Chrétien P., Bastien L., Slilaty S. N. Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli. J Biol Chem. 1991 Jan 15;266(2):1058–1063. [PubMed] [Google Scholar]
  42. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Usuda Y., Kubota A., Berk A. J., Handa H. Affinity purification of transcription factor IIA from HeLa cell nuclear extracts. EMBO J. 1991 Aug;10(8):2305–2310. doi: 10.1002/j.1460-2075.1991.tb07767.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Waldschmidt R., Seifart K. H. TFIIA is required for in vitro transcription of mammalian U6 genes by RNA polymerase III. J Biol Chem. 1992 Aug 15;267(23):16359–16364. [PubMed] [Google Scholar]
  45. Wampler S. L., Tyree C. M., Kadonaga J. T. Fractionation of the general RNA polymerase II transcription factors from Drosophila embryos. J Biol Chem. 1990 Dec 5;265(34):21223–21231. [PubMed] [Google Scholar]
  46. Wang W., Gralla J. D., Carey M. The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA. Genes Dev. 1992 Sep;6(9):1716–1727. doi: 10.1101/gad.6.9.1716. [DOI] [PubMed] [Google Scholar]
  47. Wertman K. F., Drubin D. G., Botstein D. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 1992 Oct;132(2):337–350. doi: 10.1093/genetics/132.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yokomori K., Admon A., Goodrich J. A., Chen J. L., Tjian R. Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex. Genes Dev. 1993 Nov;7(11):2235–2245. doi: 10.1101/gad.7.11.2235. [DOI] [PubMed] [Google Scholar]
  49. Zawel L., Reinberg D. Initiation of transcription by RNA polymerase II: a multi-step process. Prog Nucleic Acid Res Mol Biol. 1993;44:67–108. doi: 10.1016/s0079-6603(08)60217-2. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES