Mammalian transcription activation domains of VP16, AP2 and CTF activate transcription in a whole cell extract from Schizosaccharomyces pombe through the SRB/mediator

• Published in: Yeast (Chichester, England) England), 2005-05, Vol.22 (7), p.511-521
• Main Authors: Tamayo, Evelyn, Bernal, Giuliano, Maldonado, Edio
• Format: Article
• Language: English
• Subjects: Animals; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Gene Expression Regulation, Fungal; Humans; in vitro transcription; mediator; Mediator Complex; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA Polymerase II - metabolism; RNAPII; S. pombe; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - chemistry; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; transcription activation; Transcription Factor AP-2; transcription factors; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Transcriptional Activation
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/yea.1228

The acidic‐rich activation domain of VP16 and the proline‐rich activation domains of human AP2 and human CTF are able to activate transcription in a whole cell extract from Schizosaccharomyces pombe, whereas the glutamine‐rich domains of Sp1 and Oct2 are unable to activate transcription in this system. Immunodepletion experiments of the whole cell extracts using specific antibodies against pombe TAF110, pombe TAF 72, pombe TBP and Srb4 shows that the activation of transcription by VP16, AP2 and CTF is through the mediator, since depletion of Srb4 inhibits activated transcription but does not inhibit basal transcription. Immunodepletion of TBP causes inhibition of both activated and basal transcription. On the other hand, immunodepletion of TAFs does not have an effect on either activated or basal transcription. Purified RNA polymerase holoenzyme is able to rescue the transcriptional activation activity of the anti‐Srb4 immunodepleted extract. Moreover, we demonstrate that the mediator is needed for basal transcription of a TATA‐less promoter. Copyright © 2005 John Wiley & Sons, Ltd.