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

Abstract

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4⌬ cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

Selective role of Mediator tail module in the transcription of highly regulated genes in yeast

“…7,11,35 The Med15 subunit has also been shown to regulate galactose and amino acid metabolism in yeast by directly interacting with activators Gal4 and Gcn4. 10,[36][37][38] The requirement of Med15 in the metabolism of xenobiotics has also been reported in yeast and C. elegans. In yeast, Med15 directly binds to Pdr1 orthologs, which regulate the expression of genes involved in drug-efflux pumps.…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Over the past 10 years, a combination of biochemical, genetic, and structural experiments has conclusively identified relevant targets for some mammalian and yeast activators (e.g., Stevens et al 2002;Yang et al 2004;Green 2005;Sampietro et al 2006;Waters et al 2006;Thakur et al 2009;Herbig et al 2010;Jedidi et al 2010). In general, these targets are located in coactivator complexes and chromatin-remodeling or -modifying factors rather than in subunits of the general transcription factors.…”

“…Both activation domains contact at least three common activator-binding domains on Gal11/ Med15, each of which contributes additively to activated transcription (Herbig et al 2010). Activator-Gal11 binding has micromolar affinity and, for those sites measured, a half life of less than one millisecond (Jedidi et al 2010;Brzovic et al 2011). In this model, both Gcn4 activation domains rapidly sample the Gal11 activator-binding domains, and Mediator is recruited to the regulatory region without a stable high-affinity activator-target interaction.…”

“…Mutation of any one of these subunits disrupts this submodule. The best-characterized tail subunit is Med15/Gal11, containing four N-terminal activator-binding domains separated by glutamine or glutamine-asparaginerich flexible linkers (Herbig et al 2010;Jedidi et al 2010).…”

Mediator Recruitment to Heat Shock Genes Requires Dual Hsf1 Activation Domains and Mediator Tail Subunits Med15 and Med16

“…This conclusion follows from the fact that Hsf1 bearing just one activator (Hsf1 ⌬CTA or Hsf1 ⌬NTA ) recruited Mediator to only 5-33% of the level achieved by Hsf1 ϩ bearing two activation domains. In this regard, Hsf1 may resemble the well characterized Gcn4 activator, whose tandem activation domains target three non-contiguous segments of Med15 in a partially redundant manner (63,64) and whose N-terminal activation domain additionally targets Med16 (64). Further studies will be necessary to identify the domains within Med15 and Med16 targeted by Hsf1.…”