The Structural and Functional Role of Med5 in the Yeast Mediator Tail Module

Abstract

Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5⌬ and med15⌬ deletions. The med5⌬ and med16⌬ deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.The multiprotein Mediator complex is required for basal and regulated expression of nearly all RNA polymerase II (pol II) 3 -dependent genes in the Saccharomyces cerevisiae genome. Mediator conveys regulatory information from enhancers and other control elements to the promoter (1). The functional activities identified for Mediator include stimulation of basal transcription, support of activated transcription, and enhancement of phosphorylation of the C-terminal domain of pol II by the transcription factor IIH kinase (2, 3). S. cerevisiae Mediator also contains a histone acetyltransferase (HAT) activity, which is not found in other eukaryotic Mediator complexes (4). The HAT activity was localized to Med5 (Nut1), a S. cerevisiae-specific protein, which lacks homologues in higher eukaryotes (4, 5). The MED5 gene was originally isolated in a screen for mutants that would suppress the Swi4/Swi6 dependence of a synthetic reporter gene containing part of the HO promoter (6). Several other genes encoding Mediator proteins were identified in the same screen, including MED10 (NUT2), MED16 (SIN4), MED19 (ROX3), MED12 (SRB8), MED13 (SRB9), CDK8 (SRB10), and CYCC (SRB11). The MED5 gene is nonessential in yeast. A deletion of MED5 relieves repression at the URS2 element in the HO promoter but only in combination with a mutant allele of either MED10 or CCR4 (6). These effects on the HO promoter were seen with a lacZ reporter gene but not at the endogenous HO gene locus. The in vivo role of Med5 in Mediator-dependent gene expression therefore remains an open question.In the presence of RNA pol II, Mediator adopts an extended conformation that embraces the globular pol II core complex (7). The extended structure reveals three distinct submodules of Mediator. Direct contacts are formed between pol II and the head and middle region (7,8). The largest part of Mediator is made up of an elongated tail region, which does not appear to contact pol II. Structural analysis of mutant Mediator complex has demonstrated that the tail region contains the Med2, Med3, Med15 (Gal11), and Med16, proteins, which are involved in interactions with a number of different activators,...

A Functional Module of Yeast Mediator That Governs the Dynamic Range of Heat-Shock Gene Expression

“…Therefore, as Cdk8/Srb10 and Gal11 are encoded by nonessential genes, we constructed isogenic srb10D and gal11D derivatives of the parental strain. We also deleted SRB2, which encodes a nonessential subunit of the head domain, and NUT1, which encodes a tail domain subunit that negatively regulates the expression of oxidative phosphorylation genes (Beve et al 2005). However, as shown in Figure 4A, deletion of SRB10, GAL11, or SRB2 fails to enhance the low basal expression of hsp82-DHSE1; its induced expression is likewise unaffected ( Figure 4B).…”

“…Biochemical and electron microscopic analysis of yeast Mediator have indicated the presence of three discrete domains: the ''head,'' composed of Med6, Med8, Med11, Med17/Srb4, Med18/Srb5, Med19/ Rox3, Med20/Srb2, and Med22/Srb6; the ''middle,'' composed of Med1, Med4, Med7, Med9, Med10/Nut2, Med21/Srb7, and Med31/Soh1; and the ''tail,'' composed of Med2, Med3/Hrs1, Med5/Nut1, Med14/Rgr1, Med15/Gal11, and Med16/Sin4 (Boube et al 2002;Beve et al 2005). A kinase module, composed of Cdk8/ Srb10, CycC/Srb11, Med12/Srb8, and Med13/Srb9, loosely associates with the 21-subunit core Mediator complex (Liu et al 2001).…”

“…In addition, at several yeast promoters, recruitment of Mediator has been found to precede that of pol II (Bhoite et al 2001;Cosma et al 2001;Bryant and Ptashne 2003), demonstrating that it can exist free of RNA polymerase in vivo. Mediator has been shown to associate exclusively with the UAS of GAL genes and does so independently of pol II and GTFs (Kuras et al 2003).Biochemical and electron microscopic analysis of yeast Mediator have indicated the presence of three discrete domains: the ''head,'' composed of Med6, Med8, Med11, Med17/Srb4, Med18/Srb5, Med19/ Rox3, Med20/Srb2, and Med22/Srb6; the ''middle,'' composed of Med1, Med4, Med7, Med9, Med10/Nut2, Med21/Srb7, and Med31/Soh1; and the ''tail,'' composed of Med2, Med3/Hrs1, Med5/Nut1, Med14/Rgr1, Med15/Gal11, and Med16/Sin4 (Boube et al 2002;Beve et al 2005). A kinase module, composed of Cdk8/ Srb10, CycC/Srb11, Med12/Srb8, and Med13/Srb9, loosely associates with the 21-subunit core Mediator complex (Liu et al 2001).…”

Med19(Rox3) Regulates Intermodule Interactions in the Saccharomyces cerevisiae Mediator Complex

“…Yellow subunits are in the Tail module, green subunits are in the Middle module, and blue subunits are in the Head module. The assignment of subunits is as previously described (3,11), except for the assignment of Med5(Nut1) as a Tail subunit (41). B, an alternative model in which Med14(Rgr1) spans the Head and Tail domain and Med19(Rox3) stabilizes the interaction of Middle module subunits with the rest of the complex.…”

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

“…The tail consists of Med15/Gal11, Med16/Sin4, Med2, Med3/Pgd1/Hrs1, and Med5/Nut1, none of which is essential in vivo (21,28), and is tethered to the middle domain via the Med14/Rgr1-CTD. Deletion of Med16/Sin4 leads to loss of the remaining tail subunits from Mediator (20,29,30), and the released subcomplex containing Gal11, Med2, and Pgd1 is stable in cell extracts, capable of binding Gcn4 in vitro, and recruited independently of head subunits by Gcn4 to target genes in vivo (26).…”