Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo.
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.
Jedidi
Iness
I
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Zhang
Fan
F
Qiu
Hongfang
H
Stahl
Stephen J
SJ
Palmer
Ira
I
Kaufman
Joshua D
JD
Nadaud
Philippe S
PS
Mukherjee
Sujoy
S
Wingfield
Paul T
PT
Jaroniec
Christopher P
CP
Hinnebusch
Alan G
AG
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D015966:Gene Expression Regulation, Fungal; D056892:Mediator Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010641:Phenotype; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology
D050976:Basic-Leucine Zipper Transcription Factors; C058337:GAL11 protein, S cerevisiae; C501391:GCN4 protein, S cerevisiae; D056892:Mediator Complex; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II