Saccharomyces cerevisiae Med9 comprises two functionally distinct domains that play different roles in transcriptional regulation.
Mediator is one of the most important co-activators that function in eukaryotic transcriptional regulation. In Saccharomyces cerevisiae, Mediator is comprised of 25 subunits belonging to four structurally distinct modules: Head, Middle, Tail, and Cyc-C. Although each module plays a critical role in the regulation of a distinct set of genes, the precise molecular mechanisms remain unclear. To gain new insight into the role of the less-characterized Middle module, we analyzed the function of Med9 by constructing a set of mutants and subjecting them to a range of in vivo and in vitro assays. Our results demonstrate that Med9 has two functional domains. The species-specific amino-terminal half (aa 1-63) plays a regulatory role in transcriptional regulation in vivo and in vitro. In contrast, the well-conserved carboxy-terminal half (aa 64-149) has a more fundamental function involved in direct binding to the amino-terminal portions of Med4 and Med7 and the assembly of Med9 into the Middle module. Importantly, activator-dependent recruitment of TBP and Taf11 to the promoter is differentially affected in med9 extracts and in extracts lacking Mediator. Add-back experiments indicate that some unidentified factor(s) in med9 extracts may impact the binding of TFIID to the promoter.
Takahashi
Hiroyuki
H
Division of Molecular and Cellular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Yokohama 230-0045, Japan.
Kasahara
Koji
K
Kokubo
Tetsuro
T
D015180:Electrophoresis, Gel, Two-Dimensional; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D056892:Mediator Complex; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics
C082275:CSE2 protein, S cerevisiae; C110184:MED7 protein, S cerevisiae; D056892:Mediator Complex; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors