Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Apr;17(4):1868–1880. doi: 10.1128/mcb.17.4.1868

Functional dissection of the B" component of RNA polymerase III transcription factor IIIB: a scaffolding protein with multiple roles in assembly and initiation of transcription.

A Kumar 1, G A Kassavetis 1, E P Geiduschek 1, M Hambalko 1, C J Brent 1
PMCID: PMC232034  PMID: 9121435

Abstract

Transcription factor IIIB (TFIIIB), the central transcription factor of Saccharomyces cerevisiae RNA polymerase III, is composed of TATA-binding protein, the TFIIB-related protein Brf, and B". B", the last component to enter the TFIIIB-DNA complex, confers extremely tight DNA binding on TFIIIB. Terminally and internally deleted B" derivatives were tested for competence to form TFIIIB-DNA complexes by TFIIIC-dependent and -independent pathways on the SUP4 tRNA(Tyr) and U6 snRNA (SNR6) genes, respectively, and for transcription. Selected TFIIIB-TFIIIC-DNA complexes assembled with truncated B" were analyzed by DNase I footprinting, and the surface topography of B" in the TFIIIB-DNA complex was also analyzed by hydroxyl radical protein footprinting. These analyses define functional domains of B" and also reveal roles in start site selection by RNA polymerase III and in clearing TFIIIC from the transcriptional start. Although absolutely required for transcription, B" can be extensively truncated. Core proteins retaining as few as 176 (of 594) amino acids remain competent to transcribe the SNR6 gene in vitro. TFIIIC-dependent assembly on DNA and transcription requires a larger core of B": two domains (I and II) that are required for SNR6 transcription on an either-or basis are simultaneously required for TFIIIC-dependent assembly of DNA complexes and transcription. Domains I and II of B" are buried upon assembly of the TFIIIB-DNA complex, as determined by protein footprinting. The picture of the TFIIIB-DNA complex that emerges is that B" serves as its scaffold and is folded over in the complex so that domains I and II are near one another.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Aasland R., Stewart A. F., Gibson T. The SANT domain: a putative DNA-binding domain in the SWI-SNF and ADA complexes, the transcriptional co-repressor N-CoR and TFIIIB. Trends Biochem Sci. 1996 Mar;21(3):87–88. [PubMed] [Google Scholar]
  2. Archambault J., Milne C. A., Schappert K. T., Baum B., Friesen J. D., Segall J. The deduced sequence of the transcription factor TFIIIA from Saccharomyces cerevisiae reveals extensive divergence from Xenopus TFIIIA. J Biol Chem. 1992 Feb 15;267(5):3282–3288. [PubMed] [Google Scholar]
  3. Bartholomew B., Kassavetis G. A., Braun B. R., Geiduschek E. P. The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent. EMBO J. 1990 Jul;9(7):2197–2205. doi: 10.1002/j.1460-2075.1990.tb07389.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartholomew B., Kassavetis G. A., Geiduschek E. P. Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC. Mol Cell Biol. 1991 Oct;11(10):5181–5189. doi: 10.1128/mcb.11.10.5181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braun B. R., Bartholomew B., Kassavetis G. A., Geiduschek E. P. Topography of transcription factor complexes on the Saccharomyces cerevisiae 5 S RNA gene. J Mol Biol. 1992 Dec 20;228(4):1063–1077. doi: 10.1016/0022-2836(92)90315-b. [DOI] [PubMed] [Google Scholar]
  6. Braun B. R., Kassavetis G. A., Geiduschek E. P. Bending of the Saccharomyces cerevisiae 5S rRNA gene in transcription factor complexes. J Biol Chem. 1992 Nov 5;267(31):22562–22569. [PubMed] [Google Scholar]
  7. Braun B. R., Riggs D. L., Kassavetis G. A., Geiduschek E. P. Multiple states of protein-DNA interaction in the assembly of transcription complexes on Saccharomyces cerevisiae 5S ribosomal RNA genes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2530–2534. doi: 10.1073/pnas.86.8.2530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Buratowski S., Zhou H. A suppressor of TBP mutations encodes an RNA polymerase III transcription factor with homology to TFIIB. Cell. 1992 Oct 16;71(2):221–230. doi: 10.1016/0092-8674(92)90351-c. [DOI] [PubMed] [Google Scholar]
  10. Burnol A. F., Margottin F., Schultz P., Marsolier M. C., Oudet P., Sentenac A. Basal promoter and enhancer element of yeast U6 snRNA gene. J Mol Biol. 1993 Oct 20;233(4):644–658. doi: 10.1006/jmbi.1993.1542. [DOI] [PubMed] [Google Scholar]
  11. Chaussivert N., Conesa C., Shaaban S., Sentenac A. Complex interactions between yeast TFIIIB and TFIIIC. J Biol Chem. 1995 Jun 23;270(25):15353–15358. doi: 10.1074/jbc.270.25.15353. [DOI] [PubMed] [Google Scholar]
  12. Colbert T., Hahn S. A yeast TFIIB-related factor involved in RNA polymerase III transcription. Genes Dev. 1992 Oct;6(10):1940–1949. doi: 10.1101/gad.6.10.1940. [DOI] [PubMed] [Google Scholar]
  13. Eschenlauer J. B., Kaiser M. W., Gerlach V. L., Brow D. A. Architecture of a yeast U6 RNA gene promoter. Mol Cell Biol. 1993 May;13(5):3015–3026. doi: 10.1128/mcb.13.5.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geiduschek E. P., Kassavetis G. A. Comparing transcriptional initiation by RNA polymerases I and III. Curr Opin Cell Biol. 1995 Jun;7(3):344–351. doi: 10.1016/0955-0674(95)80089-1. [DOI] [PubMed] [Google Scholar]
  15. Greiner D. P., Hughes K. A., Gunasekera A. H., Meares C. F. Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):71–75. doi: 10.1073/pnas.93.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Hernandez N. TBP, a universal eukaryotic transcription factor? Genes Dev. 1993 Jul;7(7B):1291–1308. doi: 10.1101/gad.7.7b.1291. [DOI] [PubMed] [Google Scholar]
  18. Heyduk E., Heyduk T. Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach. Biochemistry. 1994 Aug 16;33(32):9643–9650. doi: 10.1021/bi00198a033. [DOI] [PubMed] [Google Scholar]
  19. Heyduk T., Heyduk E., Severinov K., Tang H., Ebright R. H. Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10162–10166. doi: 10.1073/pnas.93.19.10162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huet J., Conesa C., Manaud N., Chaussivert N., Sentenac A. Interactions between yeast TFIIIB components. Nucleic Acids Res. 1994 Aug 25;22(16):3433–3439. doi: 10.1093/nar/22.16.3433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huet J., Sentenac A. The TATA-binding protein participates in TFIIIB assembly on tRNA genes. Nucleic Acids Res. 1992 Dec 25;20(24):6451–6454. doi: 10.1093/nar/20.24.6451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Joazeiro C. A., Kassavetis G. A., Geiduschek E. P. Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes. Genes Dev. 1996 Mar 15;10(6):725–739. doi: 10.1101/gad.10.6.725. [DOI] [PubMed] [Google Scholar]
  23. Joazeiro C. A., Kassavetis G. A., Geiduschek E. P. Identical components of yeast transcription factor IIIB are required and sufficient for transcription of TATA box-containing and TATA-less genes. Mol Cell Biol. 1994 Apr;14(4):2798–2808. doi: 10.1128/mcb.14.4.2798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kassavetis G. A., Bartholomew B., Blanco J. A., Johnson T. E., Geiduschek E. P. Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7308–7312. doi: 10.1073/pnas.88.16.7308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kassavetis G. A., Braun B. R., Nguyen L. H., Geiduschek E. P. S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors. Cell. 1990 Jan 26;60(2):235–245. doi: 10.1016/0092-8674(90)90739-2. [DOI] [PubMed] [Google Scholar]
  26. Kassavetis G. A., Joazeiro C. A., Pisano M., Geiduschek E. P., Colbert T., Hahn S., Blanco J. A. The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. Cell. 1992 Dec 11;71(6):1055–1064. doi: 10.1016/0092-8674(92)90399-w. [DOI] [PubMed] [Google Scholar]
  27. Kassavetis G. A., Nguyen S. T., Kobayashi R., Kumar A., Geiduschek E. P., Pisano M. Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9786–9790. doi: 10.1073/pnas.92.21.9786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kassavetis G. A., Riggs D. L., Negri R., Nguyen L. H., Geiduschek E. P. Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes. Mol Cell Biol. 1989 Jun;9(6):2551–2566. doi: 10.1128/mcb.9.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Khoo B., Brophy B., Jackson S. P. Conserved functional domains of the RNA polymerase III general transcription factor BRF. Genes Dev. 1994 Dec 1;8(23):2879–2890. doi: 10.1101/gad.8.23.2879. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. Lefebvre O., Carles C., Conesa C., Swanson R. N., Bouet F., Riva M., Sentenac A. TFC3: gene encoding the B-block binding subunit of the yeast transcription factor IIIC. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10512–10516. doi: 10.1073/pnas.89.21.10512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Léveillard T., Kassavetis G. A., Geiduschek E. P. Repression and redirection of Saccharomyces cerevisiae tRNA synthesis from upstream of the transcriptional start site. J Biol Chem. 1993 Feb 15;268(5):3594–3603. [PubMed] [Google Scholar]
  35. Léveillard T., Kassavetis G. A., Geiduschek E. P. Saccharomyces cerevisiae transcription factors IIIB and IIIC bend the DNA of a tRNA(Gln) gene. J Biol Chem. 1991 Mar 15;266(8):5162–5168. [PubMed] [Google Scholar]
  36. López-De-León A., Librizzi M., Puglia K., Willis I. M. PCF4 encodes an RNA polymerase III transcription factor with homology to TFIIB. Cell. 1992 Oct 16;71(2):211–220. doi: 10.1016/0092-8674(92)90350-l. [DOI] [PubMed] [Google Scholar]
  37. Marck C., Lefebvre O., Carles C., Riva M., Chaussivert N., Ruet A., Sentenac A. The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4027–4031. doi: 10.1073/pnas.90.9.4027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Margottin F., Dujardin G., Gérard M., Egly J. M., Huet J., Sentenac A. Participation of the TATA factor in transcription of the yeast U6 gene by RNA polymerase C. Science. 1991 Jan 25;251(4992):424–426. doi: 10.1126/science.1989075. [DOI] [PubMed] [Google Scholar]
  39. Matsuzaki H., Kassavetis G. A., Geiduschek E. P. Analysis of RNA chain elongation and termination by Saccharomyces cerevisiae RNA polymerase III. J Mol Biol. 1994 Jan 28;235(4):1173–1192. doi: 10.1006/jmbi.1994.1072. [DOI] [PubMed] [Google Scholar]
  40. Parsons M. C., Weil P. A. Cloning of TFC1, the Saccharomyces cerevisiae gene encoding the 95-kDa subunit of transcription factor TFIIIC. J Biol Chem. 1992 Feb 15;267(5):2894–2901. [PubMed] [Google Scholar]
  41. Rameau G., Puglia K., Crowe A., Sethy I., Willis I. A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III. Mol Cell Biol. 1994 Jan;14(1):822–830. doi: 10.1128/mcb.14.1.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Roberts S., Colbert T., Hahn S. TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter. Genes Dev. 1995 Apr 1;9(7):832–842. doi: 10.1101/gad.9.7.832. [DOI] [PubMed] [Google Scholar]
  43. Roberts S., Miller S. J., Lane W. S., Lee S., Hahn S. Cloning and functional characterization of the gene encoding the TFIIIB90 subunit of RNA polymerase III transcription factor TFIIIB. J Biol Chem. 1996 Jun 21;271(25):14903–14909. doi: 10.1074/jbc.271.25.14903. [DOI] [PubMed] [Google Scholar]
  44. Rüth J., Conesa C., Dieci G., Lefebvre O., Düsterhöft A., Ottonello S., Sentenac A. A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB. EMBO J. 1996 Apr 15;15(8):1941–1949. [PMC free article] [PubMed] [Google Scholar]
  45. Schultz P., Marzouki N., Marck C., Ruet A., Oudet P., Sentenac A. The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy. EMBO J. 1989 Dec 1;8(12):3815–3824. doi: 10.1002/j.1460-2075.1989.tb08559.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Spolar R. S., Record M. T., Jr Coupling of local folding to site-specific binding of proteins to DNA. Science. 1994 Feb 11;263(5148):777–784. doi: 10.1126/science.8303294. [DOI] [PubMed] [Google Scholar]
  47. Starr D. B., Hoopes B. C., Hawley D. K. DNA bending is an important component of site-specific recognition by the TATA binding protein. J Mol Biol. 1995 Jul 21;250(4):434–446. doi: 10.1006/jmbi.1995.0388. [DOI] [PubMed] [Google Scholar]
  48. Swanson R. N., Conesa C., Lefebvre O., Carles C., Ruet A., Quemeneur E., Gagnon J., Sentenac A. Isolation of TFC1, a gene encoding one of two DNA-binding subunits of yeast transcription factor tau (TFIIIC). Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4887–4891. doi: 10.1073/pnas.88.11.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Werner M., Chaussivert N., Willis I. M., Sentenac A. Interaction between a complex of RNA polymerase III subunits and the 70-kDa component of transcription factor IIIB. J Biol Chem. 1993 Oct 5;268(28):20721–20724. [PubMed] [Google Scholar]
  50. Whitehall S. K., Kassavetis G. A., Geiduschek E. P. The symmetry of the yeast U6 RNA gene's TATA box and the orientation of the TATA-binding protein in yeast TFIIIB. Genes Dev. 1995 Dec 1;9(23):2974–2985. doi: 10.1101/gad.9.23.2974. [DOI] [PubMed] [Google Scholar]

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

RESOURCES