A triad of subunits from the Gal11/tail domain of Srb mediator is an in vivo target of transcriptional activator Gcn4p.
The Srb mediator is an important transcriptional coactivator for Gcn4p in the yeast Saccharomyces cerevisiae. We show that three subunits of the Gal11/tail domain of mediator, Gal11p, Pgd1p, and Med2p, and the head domain subunit Srb2p make overlapping contributions to the interaction of mediator with recombinant Gcn4p in vitro. Each of these proteins, along with the tail subunit Sin4p, also contributes to the recruitment of mediator by Gcn4p to target promoters in vivo. We found that Gal11p, Med2p, and Pgd1p reside in a stable subcomplex in sin4Delta cells that interacts with Gcn4p in vitro and that is recruited independently of the rest of mediator by Gcn4p in vivo. Thus, the Gal11p/Med2p/Pgd1p triad is both necessary for recruitment of intact mediator and appears to be sufficient for recruitment by Gcn4p as a free subcomplex. The med2Delta mutation impairs the recruitment of TATA binding protein (TBP) and RNA polymerase II to the promoter and the induction of transcription at ARG1, demonstrating the importance of the tail domain for activation by Gcn4p in vivo. Even though the Gal11p/Med2p/Pgd1p triad is the only portion of Srb mediator recruited efficiently to the promoter in the sin4Delta strain, this mutant shows high-level TBP recruitment and wild-type transcriptional induction at ARG1. Hence, the Gal11p/Med2p/Pgd1p triad may contribute to TBP recruitment independently of the rest of mediator.
Zhang
Fan
F
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Building 6A/Room B1A13, Bethesda, MD 20892, USA.
Sumibcay
Laarni
L
Hinnebusch
Alan G
AG
Swanson
Mark J
MJ
D015152:Blotting, Northern; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005838:Genotype; D005982:Glutathione Transferase / Q000378:metabolism; D056892:Mediator Complex; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic
D002843:Chromatin; D004268:DNA-Binding Proteins; C058337:GAL11 protein, S cerevisiae; C470560:MED2 protein, S cerevisiae; D056892:Mediator Complex; D009687:Nuclear Proteins; C110281:PGD1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D005982:Glutathione Transferase; D011494:Protein Kinases