Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Dec;17(12):6794–6802. doi: 10.1128/mcb.17.12.6794

Transcriptional activation by TFIIB mutants that are severely impaired in interaction with promoter DNA and acidic activation domains.

S Chou 1, K Struhl 1
PMCID: PMC232535  PMID: 9372910

Abstract

Biochemical experiments indicate that the general transcription factor IIB (TFIIB) can interact directly with acidic activation domains and that activators can stimulate transcription by increasing recruitment of TFIIB to promoters. For promoters at which recruitment of TFIIB to promoters is limiting in vivo, one would predict that transcriptional activity should be particularly sensitive to TFIIB mutations that decrease the association of TFIIB with promoter DNA and/or with activation domains; i.e., such TFIIB mutations should exacerbate a limiting step that occurs in wild-type cells. Here, we describe mutations on the DNA-binding surface of TFIIB that severely affect both TATA-binding protein (TBP)-TFIIB-TATA complex formation and interaction with the VP16 activation domain in vitro. These TFIIB mutations affect the stability of the TBP-TFIIB-TATA complex in vivo because they are synthetically lethal in combination with TBP mutants impaired for TFIIB binding. Interestingly, these TFIIB derivatives support viability, and they efficiently respond to Gal4-VP16 and natural acidic activators in different promoter contexts. These results suggest that in vivo, recruitment of TFIIB is not generally a limiting step for acidic activators. However, one TFIIB derivative shows reduced transcription of GAL4, suggesting that TFIIB may be limiting at a subset of promoters in vivo.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Apone L. M., Virbasius C. M., Reese J. C., Green M. R. Yeast TAF(II)90 is required for cell-cycle progression through G2/M but not for general transcription activation. Genes Dev. 1996 Sep 15;10(18):2368–2380. doi: 10.1101/gad.10.18.2368. [DOI] [PubMed] [Google Scholar]
  2. Arndt K. M., Ricupero-Hovasse S., Winston F. TBP mutants defective in activated transcription in vivo. EMBO J. 1995 Apr 3;14(7):1490–1497. doi: 10.1002/j.1460-2075.1995.tb07135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bangur C. S., Pardee T. S., Ponticelli A. S. Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein-TFIIB-DNA complexes. Mol Cell Biol. 1997 Dec;17(12):6784–6793. doi: 10.1128/mcb.17.12.6784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barberis A., Pearlberg J., Simkovich N., Farrell S., Reinagel P., Bamdad C., Sigal G., Ptashne M. Contact with a component of the polymerase II holoenzyme suffices for gene activation. Cell. 1995 May 5;81(3):359–368. doi: 10.1016/0092-8674(95)90389-5. [DOI] [PubMed] [Google Scholar]
  5. Berger S. L., Piña B., Silverman N., Marcus G. A., Agapite J., Regier J. L., Triezenberg S. J., Guarente L. Genetic isolation of ADA2: a potential transcriptional adaptor required for function of certain acidic activation domains. Cell. 1992 Jul 24;70(2):251–265. doi: 10.1016/0092-8674(92)90100-q. [DOI] [PubMed] [Google Scholar]
  6. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  7. Bryant G. O., Martel L. S., Burley S. K., Berk A. J. Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo. Genes Dev. 1996 Oct 1;10(19):2491–2504. doi: 10.1101/gad.10.19.2491. [DOI] [PubMed] [Google Scholar]
  8. Chatterjee S., Struhl K. Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain. Nature. 1995 Apr 27;374(6525):820–822. doi: 10.1038/374820a0. [DOI] [PubMed] [Google Scholar]
  9. Chen W., Struhl K. Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2691–2695. doi: 10.1073/pnas.85.8.2691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Colgan J., Ashali H., Manley J. L. A direct interaction between a glutamine-rich activator and the N terminus of TFIIB can mediate transcriptional activation in vivo. Mol Cell Biol. 1995 Apr;15(4):2311–2320. doi: 10.1128/mcb.15.4.2311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Colgan J., Wampler S., Manley J. L. Interaction between a transcriptional activator and transcription factor IIB in vivo. Nature. 1993 Apr 8;362(6420):549–553. doi: 10.1038/362549a0. [DOI] [PubMed] [Google Scholar]
  12. Cormack B. P., Strubin M., Ponticelli A. S., Struhl K. Functional differences between yeast and human TFIID are localized to the highly conserved region. Cell. 1991 Apr 19;65(2):341–348. doi: 10.1016/0092-8674(91)90167-w. [DOI] [PubMed] [Google Scholar]
  13. Dubrovskaya V., Lavigne A. C., Davidson I., Acker J., Staub A., Tora L. Distinct domains of hTAFII100 are required for functional interaction with transcription factor TFIIF beta (RAP30) and incorporation into the TFIID complex. EMBO J. 1996 Jul 15;15(14):3702–3712. [PMC free article] [PubMed] [Google Scholar]
  14. Fang S. M., Burton Z. F. RNA polymerase II-associated protein (RAP) 74 binds transcription factor (TF) IIB and blocks TFIIB-RAP30 binding. J Biol Chem. 1996 May 17;271(20):11703–11709. doi: 10.1074/jbc.271.20.11703. [DOI] [PubMed] [Google Scholar]
  15. Gonzalez-Couto E., Klages N., Strubin M. Synergistic and promoter-selective activation of transcription by recruitment of transcription factors TFIID and TFIIB. Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8036–8041. doi: 10.1073/pnas.94.15.8036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goodrich J. A., Hoey T., Thut C. J., Admon A., Tjian R. Drosophila TAFII40 interacts with both a VP16 activation domain and the basal transcription factor TFIIB. Cell. 1993 Nov 5;75(3):519–530. doi: 10.1016/0092-8674(93)90386-5. [DOI] [PubMed] [Google Scholar]
  17. Griggs D. W., Johnston M. Promoter elements determining weak expression of the GAL4 regulatory gene of Saccharomyces cerevisiae. Mol Cell Biol. 1993 Aug;13(8):4999–5009. doi: 10.1128/mcb.13.8.4999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Griggs D. W., Johnston M. Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8597–8601. doi: 10.1073/pnas.88.19.8597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gupta R., Emili A., Pan G., Xiao H., Shales M., Greenblatt J., Ingles C. J. Characterization of the interaction between the acidic activation domain of VP16 and the RNA polymerase II initiation factor TFIIB. Nucleic Acids Res. 1996 Jun 15;24(12):2324–2330. doi: 10.1093/nar/24.12.2324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ha I., Lane W. S., Reinberg D. Cloning of a human gene encoding the general transcription initiation factor IIB. Nature. 1991 Aug 22;352(6337):689–695. doi: 10.1038/352689a0. [DOI] [PubMed] [Google Scholar]
  21. Hisatake K., Ohta T., Takada R., Guermah M., Horikoshi M., Nakatani Y., Roeder R. G. Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8195–8199. doi: 10.1073/pnas.92.18.8195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hori R., Pyo S., Carey M. Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6047–6051. doi: 10.1073/pnas.92.13.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ingles C. J., Shales M., Cress W. D., Triezenberg S. J., Greenblatt J. Reduced binding of TFIID to transcriptionally compromised mutants of VP16. Nature. 1991 Jun 13;351(6327):588–590. doi: 10.1038/351588a0. [DOI] [PubMed] [Google Scholar]
  24. Iyer V., Struhl K. Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5208–5212. doi: 10.1073/pnas.93.11.5208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Iyer V., Struhl K. Mechanism of differential utilization of the his3 TR and TC TATA elements. Mol Cell Biol. 1995 Dec;15(12):7059–7066. doi: 10.1128/mcb.15.12.7059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Iyer V., Struhl K. Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure. EMBO J. 1995 Jun 1;14(11):2570–2579. doi: 10.1002/j.1460-2075.1995.tb07255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jiang Y. W., Stillman D. J. Involvement of the SIN4 global transcriptional regulator in the chromatin structure of Saccharomyces cerevisiae. Mol Cell Biol. 1992 Oct;12(10):4503–4514. doi: 10.1128/mcb.12.10.4503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kim T. K., Roeder R. G. Proline-rich activator CTF1 targets the TFIIB assembly step during transcriptional activation. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4170–4174. doi: 10.1073/pnas.91.10.4170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  30. Klages N., Strubin M. Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo. Nature. 1995 Apr 27;374(6525):822–823. doi: 10.1038/374822a0. [DOI] [PubMed] [Google Scholar]
  31. Koleske A. J., Young R. A. An RNA polymerase II holoenzyme responsive to activators. Nature. 1994 Mar 31;368(6470):466–469. doi: 10.1038/368466a0. [DOI] [PubMed] [Google Scholar]
  32. Lee M., Struhl K. A severely defective TATA-binding protein-TFIIB interaction does not preclude transcriptional activation in vivo. Mol Cell Biol. 1997 Mar;17(3):1336–1345. doi: 10.1128/mcb.17.3.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lee M., Struhl K. Mutations on the DNA-binding surface of TATA-binding protein can specifically impair the response to acidic activators in vivo. Mol Cell Biol. 1995 Oct;15(10):5461–5469. doi: 10.1128/mcb.15.10.5461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lee S., Hahn S. Model for binding of transcription factor TFIIB to the TBP-DNA complex. Nature. 1995 Aug 17;376(6541):609–612. doi: 10.1038/376609a0. [DOI] [PubMed] [Google Scholar]
  35. Li Y., Flanagan P. M., Tschochner H., Kornberg R. D. RNA polymerase II initiation factor interactions and transcription start site selection. Science. 1994 Feb 11;263(5148):805–807. doi: 10.1126/science.8303296. [DOI] [PubMed] [Google Scholar]
  36. Lin Y. S., Green M. R. Mechanism of action of an acidic transcriptional activator in vitro. Cell. 1991 Mar 8;64(5):971–981. doi: 10.1016/0092-8674(91)90321-o. [DOI] [PubMed] [Google Scholar]
  37. Lin Y. S., Ha I., Maldonado E., Reinberg D., Green M. R. Binding of general transcription factor TFIIB to an acidic activating region. Nature. 1991 Oct 10;353(6344):569–571. doi: 10.1038/353569a0. [DOI] [PubMed] [Google Scholar]
  38. Moqtaderi Z., Bai Y., Poon D., Weil P. A., Struhl K. TBP-associated factors are not generally required for transcriptional activation in yeast. Nature. 1996 Sep 12;383(6596):188–191. doi: 10.1038/383188a0. [DOI] [PubMed] [Google Scholar]
  39. Nerlov C., Ziff E. B. CCAAT/enhancer binding protein-alpha amino acid motifs with dual TBP and TFIIB binding ability co-operate to activate transcription in both yeast and mammalian cells. EMBO J. 1995 Sep 1;14(17):4318–4328. doi: 10.1002/j.1460-2075.1995.tb00106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nikolov D. B., Chen H., Halay E. D., Usheva A. A., Hisatake K., Lee D. K., Roeder R. G., Burley S. K. Crystal structure of a TFIIB-TBP-TATA-element ternary complex. Nature. 1995 Sep 14;377(6545):119–128. doi: 10.1038/377119a0. [DOI] [PubMed] [Google Scholar]
  41. Orphanides G., Lagrange T., Reinberg D. The general transcription factors of RNA polymerase II. Genes Dev. 1996 Nov 1;10(21):2657–2683. doi: 10.1101/gad.10.21.2657. [DOI] [PubMed] [Google Scholar]
  42. Ozer J., Moore P. A., Bolden A. H., Lee A., Rosen C. A., Lieberman P. M. Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription. Genes Dev. 1994 Oct 1;8(19):2324–2335. doi: 10.1101/gad.8.19.2324. [DOI] [PubMed] [Google Scholar]
  43. Pinto I., Ware D. E., Hampsey M. The yeast SUA7 gene encodes a homolog of human transcription factor TFIIB and is required for normal start site selection in vivo. Cell. 1992 Mar 6;68(5):977–988. doi: 10.1016/0092-8674(92)90040-j. [DOI] [PubMed] [Google Scholar]
  44. Ptashne M., Gann A. Transcriptional activation by recruitment. Nature. 1997 Apr 10;386(6625):569–577. doi: 10.1038/386569a0. [DOI] [PubMed] [Google Scholar]
  45. Roberts S. G., Choy B., Walker S. S., Lin Y. S., Green M. R. A role for activator-mediated TFIIB recruitment in diverse aspects of transcriptional regulation. Curr Biol. 1995 May 1;5(5):508–516. doi: 10.1016/s0960-9822(95)00103-5. [DOI] [PubMed] [Google Scholar]
  46. Roberts S. G., Green M. R. Activator-induced conformational change in general transcription factor TFIIB. Nature. 1994 Oct 20;371(6499):717–720. doi: 10.1038/371717a0. [DOI] [PubMed] [Google Scholar]
  47. Roberts S. G., Ha I., Maldonado E., Reinberg D., Green M. R. Interaction between an acidic activator and transcription factor TFIIB is required for transcriptional activation. Nature. 1993 Jun 24;363(6431):741–744. doi: 10.1038/363741a0. [DOI] [PubMed] [Google Scholar]
  48. Roeder R. G. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem Sci. 1996 Sep;21(9):327–335. [PubMed] [Google Scholar]
  49. Ruppert S., Tjian R. Human TAFII250 interacts with RAP74: implications for RNA polymerase II initiation. Genes Dev. 1995 Nov 15;9(22):2747–2755. doi: 10.1101/gad.9.22.2747. [DOI] [PubMed] [Google Scholar]
  50. Shaw S. P., Wingfield J., Dorsey M. J., Ma J. Identifying a species-specific region of yeast TF11B in vivo. Mol Cell Biol. 1996 Jul;16(7):3651–3657. doi: 10.1128/mcb.16.7.3651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stargell L. A., Struhl K. A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo. Mol Cell Biol. 1996 Aug;16(8):4456–4464. doi: 10.1128/mcb.16.8.4456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stargell L. A., Struhl K. The TBP-TFIIA interaction in the response to acidic activators in vivo. Science. 1995 Jul 7;269(5220):75–78. doi: 10.1126/science.7604282. [DOI] [PubMed] [Google Scholar]
  53. Stringer K. F., Ingles C. J., Greenblatt J. Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature. 1990 Jun 28;345(6278):783–786. doi: 10.1038/345783a0. [DOI] [PubMed] [Google Scholar]
  54. Struhl K. Chromatin structure and RNA polymerase II connection: implications for transcription. Cell. 1996 Jan 26;84(2):179–182. doi: 10.1016/s0092-8674(00)80970-8. [DOI] [PubMed] [Google Scholar]
  55. Tang H., Sun X., Reinberg D., Ebright R. H. Protein-protein interactions in eukaryotic transcription initiation: structure of the preinitiation complex. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1119–1124. doi: 10.1073/pnas.93.3.1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tansey W. P., Herr W. Selective use of TBP and TFIIB revealed by a TATA-TBP-TFIIB array with altered specificity. Science. 1997 Feb 7;275(5301):829–831. doi: 10.1126/science.275.5301.829. [DOI] [PubMed] [Google Scholar]
  57. Tansey W. P., Herr W. The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10550–10554. doi: 10.1073/pnas.92.23.10550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Walker S. S., Reese J. C., Apone L. M., Green M. R. Transcription activation in cells lacking TAFIIS. Nature. 1996 Sep 12;383(6596):185–188. doi: 10.1038/383185a0. [DOI] [PubMed] [Google Scholar]
  59. Wu Y., Reece R. J., Ptashne M. Quantitation of putative activator-target affinities predicts transcriptional activating potentials. EMBO J. 1996 Aug 1;15(15):3951–3963. [PMC free article] [PubMed] [Google Scholar]
  60. Xiao H., Friesen J. D., Lis J. T. Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator. Mol Cell Biol. 1995 Oct;15(10):5757–5761. doi: 10.1128/mcb.15.10.5757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Xiao H., Pearson A., Coulombe B., Truant R., Zhang S., Regier J. L., Triezenberg S. J., Reinberg D., Flores O., Ingles C. J. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol. 1994 Oct;14(10):7013–7024. doi: 10.1128/mcb.14.10.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yamashita S., Hisatake K., Kokubo T., Doi K., Roeder R. G., Horikoshi M., Nakatani Y. Transcription factor TFIIB sites important for interaction with promoter-bound TFIID. Science. 1993 Jul 23;261(5120):463–466. doi: 10.1126/science.8332911. [DOI] [PubMed] [Google Scholar]

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

RESOURCES