Saccharomyces cerevisiae Med9 comprises two functionally distinct domains that play different roles in transcriptional regulation

Abstract

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.

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation

“…New strains used in this work are summarized in Supplementary Table 2 ) and were constructed as follows: YTK12803, YTK13412, YTK13413, YTK13414, YTK13415, YTK13416, YTK13417 and YTK13444 were generated from YTK11411 by replacing the URA3 -marked plasmid (pYN1/ TAF1 ) with HIS3 -marked plasmids; pM7121/ taf1-ΔTAND , pM7280/ taf1-E60A E62A , pM7281/ taf1-E60A E62A D66A , pM7282/ taf1-E58A D59A , pM7283/ taf1- E58A D59A E60A E62A , pM7284/ taf1-H50A , pM7285/ taf1-H50G and pM7298/ taf1-ΔTAND2 , respectively, using a plasmid shuffle technique. Similarly, YTK13428, YTK13429, YTK13430, YTK13431, YTK13432, YTK13433, YTK13434, YTK13435, YTK13436 and YTK13536 were generated from YTK11411 by replacing pYN1/ TAF1 with LEU2 -marked plasmids; pM7118/ TAF1 55 , pM7119/ taf1-ΔTAND , pM7286/ taf1-ΔTAND1 , pM7287/ taf1-ΔTAND1 E60A E62A , pM7288/ taf1-ΔTAND1 E60A E62A D66A , pM7289/ taf1-ΔTAND1 E58A D59A , pM7290/ taf1-ΔTAND1 E58A D59A E60A E62A , pM7291/ taf1-ΔTAND1 H50A , pM7292/ taf1-ΔTAND1 H50G and pM7320/ taf1-ΔTAND1 F57A , respectively.…”

“…The 5.1 kb Not I- Sal I fragment from pM7121/ taf1-ΔTAND was ligated into the Not I/ Sal I sites of pRS315 57 to generate pM7119. pM7286/ taf1-ΔTAND1 was created by replacing the 3.4 kb Not I- Xba I fragment of pM7118/ TAF1 55 with the 3.3 kb Not I- Xba I fragment of pM7279/ taf1-ΔTAND1 . The 2.2 kb BssH II- Xba I fragments including the mutated TAND2 region were amplified by PCR from pM7280, pM7281, pM7282, pM7283, pM7284, pM7285 and pM7319 using the primer pair TK43-TK125, and then ligated into the BssH II/ Xba I sites of pM7286 to generate pM7287, pM7288, pM7289, pM7290, pM7291, pM7292 and pM7320, respectively.…”

High-throughput discovery and characterization of human transcriptional effectors

“…Several hits were not sourced from sequence-specific transcription factors where classical activator domains are expected but were instead nonclassical activators from co-activator and transcriptional machinery proteins including Med9, TFIIEβ, and NCOA3. In particular, the Med9 domain, whose ortholog directly binds other mediator complex components in yeast (Takahashi et al, 2009), was a strong activator with an average log 2 (OFF:ON) = -5.5, despite its weak expression level (Table S4). Nonclassical activators have previously been reported to work individually in yeast (Gaudreau et al, 1999) but only weakly when individually recruited in mammalian cells (Nevado et al, 1999).…”

Evolution of disorder in Mediator complex and its functional relevance

“…The interaction between these two subunits already reported in yeast appears to be crucial for the structural integrity of the Mediator complex as ScMed4 and ScMed9 interact with most other Middle module subunits. Deletion of ScMed9 affects the modular architecture of the complex ( 136 ). The interaction between Med7 and Med21, and their MoRFs are conserved between yeast and Arabidopsis .…”