ABSTRACT
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.
Subject(s)
Endopeptidases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , TATA-Box Binding Protein/metabolism , Transcriptional Activation , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Endopeptidases/genetics , Gene Expression Regulation, Fungal , Promoter Regions, Genetic , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , TATA-Box Binding Protein/genetics , UbiquitinationABSTRACT
The TBP (TATA-box-binding protein), Tbp1p, plays a vital role in all three classes of transcription by RNA polymerases I-III. A TBP1(E186D) mutation had been described that affected interaction of Tbp1p with TFIIB (transcription factor IIB) and that caused slow-growth, temperature-sensitivity, 3-aminotriazole-sensitivity as well as a gal(-) phenotype. We used the TBP1(E186D) mutant for suppressor screens, and we isolated TFIIB/SUA7(E202G) as an allele-specific suppressor of all phenotypes caused by the TBP1(E186D) mutation. Our results show that the SUA7(E202G) mutation restored binding of TFIIB to Tbp1(E186D)p. In addition, we observed that Tbp1(E186D)p was expressed at a lower level than wild-type Tbp1p, and that SUA7(E202G) restored the protein level of Tbp1(E186D)p. This suggested that the TBP1(E186D) mutation might have generated its phenotypes by making Tbp1p the limiting factor for activated transcription. DNA microarray analysis indicated that the TBP1(E186D) temperature-sensitivity and slow-growth phenotypes might have been caused by insufficient amounts of Tbp1p for efficient transcription of the rRNA genes by RNA polymerase I.
Subject(s)
Alleles , Aspartic Acid/metabolism , Cell Cycle Proteins/metabolism , Down-Regulation , Glutamic Acid/metabolism , Glycine/metabolism , Saccharomyces cerevisiae Proteins/metabolism , TATA-Box Binding Protein/metabolism , Transcription Factor TFIIB/metabolism , Aspartic Acid/genetics , Basic-Leucine Zipper Transcription Factors , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Glutamic Acid/genetics , Glycine/genetics , Mutation/genetics , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , TATA-Box Binding Protein/genetics , Temperature , Transcription Factor TFIIB/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
According to the recruitment model, transcriptional activators work by increasing the local concentration of one or several limiting factors for the transcription process at the target promoter. The TATA-binding protein Tbp1 has been considered as a likely candidate for such a limiting factor. We have used a series of Gal4p and Tbp1 mutants to correlate the in vivo interaction between the two proteins with the strength of activation. We find a clear correlation between activation strength and in vivo interaction for the series of Gal4p mutants. Consistently, the weaker activator Gcn4p does not interact with Tbp1. However, a corresponding analysis of the series of Tbp1 mutants revealed that Tbp1 is not an essential target of the acidic activators Gal4p and Gcn4p. Furthermore, detailed analysis of a Tbp1 mutant deficient for transcriptional activation by Gal4p revealed that the mutant is defective in interactions with five other proteins involved in the process of transcription.
