Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3.
Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)-Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.
Chew
Boon Shang
BS
Department of Microbiology, National University of Singapore, Singapore.
Siew
Wee Leng
WL
Xiao
Benjamin
B
Lehming
Norbert
N
D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D054875:Ubiquitination
D050976:Basic-Leucine Zipper Transcription Factors; C501391:GCN4 protein, S cerevisiae; C115017:SPT15 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D010450:Endopeptidases; C101111:UBP3 protein, S cerevisiae