Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo

J Biol Chem. 2010 Jan 22;285(4):2438-55. doi: 10.1074/jbc.M109.071589. Epub 2009 Nov 23.

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 sin4Delta 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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Basic-Leucine Zipper Transcription Factors* / chemistry
  • Basic-Leucine Zipper Transcription Factors* / genetics
  • Basic-Leucine Zipper Transcription Factors* / metabolism
  • Conserved Sequence
  • Gene Expression Regulation, Fungal*
  • Mediator Complex* / chemistry
  • Mediator Complex* / genetics
  • Mediator Complex* / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Phenotype
  • Protein Interaction Domains and Motifs / physiology
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • RNA Polymerase II / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins* / chemistry
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae Proteins* / metabolism
  • Transcription, Genetic / physiology

Substances

  • Basic-Leucine Zipper Transcription Factors
  • GAL11 protein, S cerevisiae
  • GCN4 protein, S cerevisiae
  • Mediator Complex
  • Protein Subunits
  • Saccharomyces cerevisiae Proteins
  • RNA Polymerase II