The F Box Protein Dsg1/Mdm30 Is a Transcriptional Coactivator that Stimulates Gal4 Turnover and Cotranscriptional mRNA Processing

Abstract

We report here that the prototypical yeast transcription factor Gal4 undergoes two distinct modes of ubiquitin-mediated proteolysis: one that occurs independent of transcription and restricts Gal4 function, and another that is transcription coupled and essential for productive activation of Gal4 target genes. Destruction of transcriptionally active Gal4 depends on an F box protein called Dsg1/Mdm30. In the absence of Dsg1, Gal4 is stable, nonubiquitylated, and unable to productively stimulate transcription. Analysis of the phenotype of dsg1-null yeast reveals a striking disconnect between GAL gene RNA and protein levels; in the absence of Dsg1, Gal4 target genes are transcribed, but the resulting RNAs are not translated. The translational defects of these RNAs are related to defects in phosphorylation of the RNA polymerase II carboxy-terminal domain, which in turn affects recruitment of RNA processing machinery. We propose that Gal4 ubiquitylation and destruction are required for initiation-competent transcription complexes to transition to fully mature elongating complexes capable of appropriate mRNA processing.

The 26S proteasome system degrades the ERM transcription factor and regulates its transcription-enhancing activity

“…In this view, polyubiquitinated forms might be targeted for degradation, whereas a transient state of mono-ubiquitination might regulate protein activity before its targeting for degradation. Ubiquitination has been linked to the regulation of the activity of some transcription factors (Bres et al, 2003;Greer et al, 2003;Peloponese et al, 2004;Muratani et al, 2005). Furthermore, ubiquitination is reported to be essential to the activity of the TADs of the VP16 and Tat proteins (Salghetti et al, 2001;Bres et al, 2003), and ubiquitination of these TADs has been shown to increase their interaction with the positive transcription elongation factor b (P-TEFb), thus augmenting the rate of transcription elongation (Kurosu and Peterlin, 2004).…”

SCFCdc4-mediated Degradation of the Hac1p Transcription Factor Regulates the Unfolded Protein Response inSaccharomyces cerevisiae

“…Finally, SCF Cdc4 -mediated degradation of Gcn4 illustrates the "OR" mode of control because it requires CPD phosphorylation by either Srb10 or Pho85 (Meimoun et al, 2000;Chi et al, 2001). Recently, another OR mode, involving a protein having two or more degrons that are regulated by different SCF complexes, has been described for Gal4p (Muratani et al, 2005). In this case, SCF Grr1 facilitates turnover of Gal4p at a relatively slow rate during yeast cell growth in the presence of a noninducing carbon source, whereas SCF Dsg1/Mdm30 mediates rapid degradation of a differentially phosphorylated form of Gal4p during growth in the presence of galactose (Muratani et al, 2005).…”

“…Detection of wild-type Hac1p and the various mutant Hac1p proteins was performed using a polyclonal anti-Hac1p antibody (Kawahara et al, 1997) or an anti-Hac1p i tail antibody . Analysis of Ura3/HA and Hac1/HA fusion proteins used anti-hemagglutinin (HA) tag antibodies, (Muratani et al, 2005), whereas detection of green fluorescent protein (GFP) fusion proteins used polyclonal anti GFP antibodies (a gift from Pamela Silver, Dana-Farber Institute, Boston, MA). Subsequently, the membranes were probed with monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (a gift from Trevor Lithgow, University of Melbourne, Melbourne, Australia) as a control for protein loading.…”

“…Unless otherwise indicated protein extracts from yeast cells were made in EZ buffer (60 mM Tris, pH 6.8, 10% [vol/vol] glycerol, 2% [wt/vol] SDS, and 5% [vol/vol] ␤-mercaptoethanol) by boiling for 10 min, followed by mixing and centrifugation (13,000 rpm for 10 min) (Muratani et al, 2005). Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA).…”

“…Recently, another OR mode, involving a protein having two or more degrons that are regulated by different SCF complexes, has been described for Gal4p (Muratani et al, 2005). In this case, SCF Grr1 facilitates turnover of Gal4p at a relatively slow rate during yeast cell growth in the presence of a noninducing carbon source, whereas SCF Dsg1/Mdm30 mediates rapid degradation of a differentially phosphorylated form of Gal4p during growth in the presence of galactose (Muratani et al, 2005). Regulation of Hac1p degradation does not involve the AND or nanoswitch modes, because it contains only a single CPD that does not require simultaneous activation by more than one kinase, although as discussed above, phosphorylation of a second nearby site may stimulate the interaction of the CPD with Cdc4p.…”