Abstract
A yeast chimeric RNA polymerase III transcription system was constructed to explore the ordered, multistep process of gene activation in vivo. A promoter-deficient U6 RNA gene harboring GAL4-binding sites could be reactivated by fusing the GAL4 DNA-binding domain to components of the general transcription factor TFIIIC (tau) or TFIIIB. Expression of chimeric tau 138 or tau 131 (but not tau 95) subunits activated transcription from GAL4-binding sites located at various positions, including upstream of or within the gene. The function(s) of the B block binding domain of TFIIIC was provided by the fused GAL4-(1-147) domain. The GAL4-(1-147)-TFIIIB70 fusion protein acted at a distance like an activator of transcription. In contrast, none of the 10 different GAL4-(1-147)-polymerase subunit fusions was able to induce transcription, suggesting that RNA polymerase recruitment is not sufficient to initiate transcription.
Full text
PDF![11938](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a9a/45351/f6543f277a1c/pnas01147-0179.png)
![11939](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a9a/45351/f8cb6988b08f/pnas01147-0180.png)
![11940](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a9a/45351/8144bd7b1f90/pnas01147-0181.png)
![11941](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a9a/45351/63bf7c6b4c43/pnas01147-0182.png)
![11942](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a9a/45351/5603c6b9268b/pnas01147-0183.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- 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]
- 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]
- Brent R., Ptashne M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell. 1985 Dec;43(3 Pt 2):729–736. doi: 10.1016/0092-8674(85)90246-6. [DOI] [PubMed] [Google Scholar]
- 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]
- Burnol A. F., Margottin F., Huet J., Almouzni G., Prioleau M. N., Méchali M., Sentenac A. TFIIIC relieves repression of U6 snRNA transcription by chromatin. Nature. 1993 Apr 1;362(6419):475–477. doi: 10.1038/362475a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Chalker D. L., Sandmeyer S. B. Ty3 integrates within the region of RNA polymerase III transcription initiation. Genes Dev. 1992 Jan;6(1):117–128. doi: 10.1101/gad.6.1.117. [DOI] [PubMed] [Google Scholar]
- 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]
- Fortner D. M., Troy R. G., Brow D. A. A stem/loop in U6 RNA defines a conformational switch required for pre-mRNA splicing. Genes Dev. 1994 Jan;8(2):221–233. doi: 10.1101/gad.8.2.221. [DOI] [PubMed] [Google Scholar]
- Gabrielsen O. S., Marzouki N., Ruet A., Sentenac A., Fromageot P. Two polypeptide chains in yeast transcription factor tau interact with DNA. J Biol Chem. 1989 May 5;264(13):7505–7511. [PubMed] [Google Scholar]
- Gabrielsen O. S., Sentenac A. RNA polymerase III (C) and its transcription factors. Trends Biochem Sci. 1991 Nov;16(11):412–416. doi: 10.1016/0968-0004(91)90166-s. [DOI] [PubMed] [Google Scholar]
- Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Lalo D., Carles C., Sentenac A., Thuriaux P. Interactions between three common subunits of yeast RNA polymerases I and III. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5524–5528. doi: 10.1073/pnas.90.12.5524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Ma J., Ptashne M. A new class of yeast transcriptional activators. Cell. 1987 Oct 9;51(1):113–119. doi: 10.1016/0092-8674(87)90015-8. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Morse R. H. Nucleosome disruption by transcription factor binding in yeast. Science. 1993 Dec 3;262(5139):1563–1566. doi: 10.1126/science.8248805. [DOI] [PubMed] [Google Scholar]
- Ostrander E. A., Benedetti P., Wang J. C. Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase. Science. 1990 Sep 14;249(4974):1261–1265. doi: 10.1126/science.2399463. [DOI] [PubMed] [Google Scholar]
- Pikaard C. S. Ribosomal gene promoter domains can function as artificial enhancers of RNA polymerase I transcription, supporting a promoter origin for natural enhancers in Xenopus. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):464–468. doi: 10.1073/pnas.91.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Werner M., Hermann-Le Denmat S., Treich I., Sentenac A., Thuriaux P. Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association. Mol Cell Biol. 1992 Mar;12(3):1087–1095. doi: 10.1128/mcb.12.3.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]