This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Woontner, M Articles by Jaehning, J A Search for Related Content PubMed PubMed Citation Articles by Woontner, M Articles by Jaehning, J A

Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

Department of Biology, Indiana University, Bloomington 47405.

ABSTRACT

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

This article has been cited by other articles:

• Shi, H., Fan, X., Sevilimedu, A., Lis, J. T. (2007). RNA aptamers directed to discrete functional sites on a single protein structural domain. Proc. Natl. Acad. Sci. USA 104: 3742-3746 [Abstract] [Full Text]  
• Garbett, K. A., Tripathi, M. K., Cencki, B., Layer, J. H., Weil, P. A. (2007). Yeast TFIID Serves as a Coactivator for Rap1p by Direct Protein-Protein Interaction. Mol. Cell. Biol. 27: 297-311 [Abstract] [Full Text]  
• Zhao, X., Shi, H., Sevilimedu, A., Liachko, N., Nelson, H. C. M., Lis, J. T. (2006). An RNA aptamer that interferes with the DNA binding of the HSF transcription activator. Nucleic Acids Res 34: 3755-3761 [Abstract] [Full Text]  
• Takagi, Y., Kornberg, R. D. (2006). Mediator as a General Transcription Factor. J. Biol. Chem. 281: 80-89 [Abstract] [Full Text]  
• Fan, X., Shi, H., Adelman, K., Lis, J. T. (2004). Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes. Proc. Natl. Acad. Sci. USA 101: 6934-6939 [Abstract] [Full Text]  
• Iida, T., Araki, H. (2004). Noncompetitive Counteractions of DNA Polymerase {varepsilon} and ISW2/yCHRAC for Epigenetic Inheritance of Telomere Position Effect in Saccharomyces cerevisiae. Mol. Cell. Biol. 24: 217-227 [Abstract] [Full Text]  
• Rondon, A. G., Jimeno, S., Garcia-Rubio, M., Aguilera, A. (2003). Molecular Evidence That the Eukaryotic THO/TREX Complex Is Required for Efficient Transcription Elongation. J. Biol. Chem. 278: 39037-39043 [Abstract] [Full Text]  
• Tan, Q., Prysak, M. H., Woychik, N. A. (2003). Loss of the Rpb4/Rpb7 Subcomplex in a Mutant Form of the Rpb6 Subunit Shared by RNA Polymerases I, II, and III. Mol. Cell. Biol. 23: 3329-3338 [Abstract] [Full Text]  
• Swanson, M. J., Qiu, H., Sumibcay, L., Krueger, A., Kim, S.-j., Natarajan, K., Yoon, S., Hinnebusch, A. G. (2003). A Multiplicity of Coactivators Is Required by Gcn4p at Individual Promoters In Vivo. Mol. Cell. Biol. 23: 2800-2820 [Abstract] [Full Text]  
• Klein, J., Nolden, M., Sanders, S. L., Kirchner, J., Weil, P. A., Melcher, K. (2003). Use of a Genetically Introduced Cross-linker to Identify Interaction Sites of Acidic Activators within Native Transcription Factor IID and SAGA. J. Biol. Chem. 278: 6779-6786 [Abstract] [Full Text]  
• Deluen, C., James, N., Maillet, L., Molinete, M., Theiler, G., Lemaire, M., Paquet, N., Collart, M. A. (2002). The Ccr4-Not Complex and yTAF1 (yTafII130p/yTafII145p) Show Physical and Functional Interactions. Mol. Cell. Biol. 22: 6735-6749 [Abstract] [Full Text]  
• Cang, Y., Prelich, G. (2002). Direct stimulation of transcription by negative cofactor 2 (NC2) through TATA-binding protein (TBP). Proc. Natl. Acad. Sci. USA 99: 12727-12732 [Abstract] [Full Text]  
• Terrell, A. R., Wongwisansri, S., Pilon, J. L., Laybourn, P. J. (2002). Reconstitution of Nucleosome Positioning, Remodeling, Histone Acetylation, and Transcriptional Activation on the PHO5 Promoter. J. Biol. Chem. 277: 31038-31047 [Abstract] [Full Text]  
• Keogh, M.-C., Cho, E.-J., Podolny, V., Buratowski, S. (2002). Kin28 Is Found within TFIIH and a Kin28-Ccl1-Tfb3 Trimer Complex with Differential Sensitivities to T-Loop Phosphorylation. Mol. Cell. Biol. 22: 1288-1297 [Abstract] [Full Text]  
• Durso, R. J., Fisher, A. K., Albright-Frey, T. J., Reese, J. C. (2001). Analysis of TAF90 Mutants Displaying Allele-Specific and Broad Defects in Transcription. Mol. Cell. Biol. 21: 7331-7344 [Abstract] [Full Text]  
• Bhaumik, S. R., Green, M. R. (2001). SAGA is an essential in vivo target of the yeast acidic activator Gal4p. Genes Dev. 15: 1935-1945 [Abstract] [Full Text]  
• Zhou, D., Lobo-Ruppert, S. M. (2001). Transcription of the Schizosaccharomyces pombe U2 gene in vivo and in vitro is directed by two essential promoter elements. Nucleic Acids Res 29: 2003-2011 [Abstract] [Full Text]  
• Mencía, M., Struhl, K. (2001). Region of Yeast TAF 130 Required for TFIID To Associate with Promoters. Mol. Cell. Biol. 21: 1145-1154 [Abstract] [Full Text]  
• Tan, Q., Li, X., Sadhale, P. P., Miyao, T., Woychik, N. A. (2000). Multiple Mechanisms of Suppression Circumvent Transcription Defects in an RNA Polymerase Mutant. Mol. Cell. Biol. 20: 8124-8133 [Abstract] [Full Text]  
• Zhang, D.-Y., Dorsey, M. J., Voth, W. P., Carson, D. J., Zeng, X., Stillman, D. J., Ma, J. (2000). Intramolecular interaction of yeast TFIIB in transcription control. Nucleic Acids Res 28: 1913-1920 [Abstract] [Full Text]  
• Donaldson, I. M., Friesen, J. D. (2000). Zinc Stoichiometry of Yeast RNA Polymerase II and Characterization of Mutations in the Zinc-binding Domain of the Largest Subunit. J. Biol. Chem. 275: 13780-13788 [Abstract] [Full Text]  
• Matangkasombut, O., Buratowski, R. M., Swilling, N. W., Buratowski, S. (2000). Bromodomain factor 1 corresponds to a missing piece of yeast TFIID. Genes Dev. 14: 951-962 [Abstract] [Full Text]  
• Tan, Q., Linask, K. L., Ebright, R. H., Woychik, N. A. (2000). Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria. Genes Dev. 14: 339-348 [Abstract] [Full Text]  
• Cho, E.-J., Buratowski, S. (1999). Evidence That Transcription Factor IIB Is Required for a Post-assembly Step in Transcription Initiation. J. Biol. Chem. 274: 25807-25813 [Abstract] [Full Text]  
• Chang, M., French-Cornay, D., Fan, H.-y., Klein, H., Denis, C. L., Jaehning, J. A. (1999). A Complex Containing RNA Polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p Plays a Role in Protein Kinase C Signaling. Mol. Cell. Biol. 19: 1056-1067 [Abstract] [Full Text]  
• Miyao, T., Woychik, N. A. (1998). RNA polymerase subunit RPB5 plays a role in transcriptional activation. Proc. Natl. Acad. Sci. USA 95: 15281-15286 [Abstract] [Full Text]  
• Komarnitsky, P. B., Klebanow, E. R., Weil, P. A., Denis, C. L. (1998). ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex. Mol. Cell. Biol. 18: 5861-5867 [Abstract] [Full Text]  
• Hampsey, M. (1998). Molecular Genetics of the RNA Polymerase II General Transcriptional Machinery. Microbiol. Mol. Biol. Rev. 62: 465-503 [Abstract] [Full Text]  
• Drysdale, C. M., Jackson, B. M., McVeigh, R., Klebanow, E. R., Bai, Y., Kokubo, T., Swanson, M., Nakatani, Y., Weil, P. A., Hinnebusch, A. G. (1998). The Gcn4p Activation Domain Interacts Specifically In Vitro with RNA Polymerase II Holoenzyme, TFIID, and the Adap-Gcn5p Coactivator Complex. Mol. Cell. Biol. 18: 1711-1724 [Abstract] [Full Text]  
• Cho, E.-J., Takagi, T., Moore, C. R., Buratowski, S. (1997). mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 11: 3319-3326 [Abstract] [Full Text]  
• Ford, J., McEwan, I. J., Wright, A. P. H., Gustafsson, J.-A. (1997). Involvement of the Transcription Factor IID Protein Complex in Gene Activation by the N-Terminal Transactivation Domain of the Glucocorticoid Receptor in Vitro. Mol. Endocrinol. 11: 1467-1475 [Abstract] [Full Text]  
• Klebanow, E. R., Poon, D., Zhou, S., Weil, P. A. (1997). Cloning and Characterization of an Essential Saccharomyces cerevisiae Gene, TAF40, Which Encodes yTAFII40, an RNA Polymerase II-specific TATA-binding Protein-associated Factor. J. Biol. Chem. 272: 9436-9442 [Abstract] [Full Text]  
• Emili, A., Ingles, C. J. (1995). Promoter-dependent Photocross-linking of the Acidic Transcriptional Activator E2F-1 to the TATA-binding Protein. J. Biol. Chem. 270: 13674-13680 [Abstract] [Full Text]  
• Guzder, S. N., Bailly, Vér., Sung, P., Prakash, L., Prakash, S. (1995). Yeast DNA Repair Protein RAD23 Promotes Complex Formation between Transcription Factor TFIIH and DNA Damage Recognition Factor RAD14. J. Biol. Chem. 270: 8385-8388 [Abstract] [Full Text]  
• Schneider, K., Smith, R., O'Shea, E. (1994). Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science 266: 122-126 [Abstract]  
• Peter, M, Herskowitz, I (1994). Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Far1. Science 265: 1228-1231 [Abstract]  
• Lorch, Y, LaPointe, J W, Kornberg, R D (1992). Initiation on chromatin templates in a yeast RNA polymerase II transcription system.. Genes Dev. 6: 2282-2287 [Abstract]  
• Lemaire, M., Collart, M. A. (2000). The TATA-binding Protein-associated Factor yTafII19p Functionally Interacts with Components of the Global Transcriptional Regulator Ccr4-Not Complex and Physically Interacts with the Not5 Subunit. J. Biol. Chem. 275: 26925-26934 [Abstract] [Full Text]  
• Orlicky, S. M., Tran, P. T., Sayre, M. H., Edwards, A. M. (2001). Dissociable Rpb4-Rpb7 Subassembly of RNA Polymerase II Binds to Single-strand Nucleic Acid and Mediates a Post-recruitment Step in Transcription Initiation. J. Biol. Chem. 276: 10097-10102 [Abstract] [Full Text]