ADA1, a Novel Component of the ADA/GCN5 Complex, Has Broader Effects than GCN5, ADA2, or ADA3

Abstract

The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here we describe the cloning, sequencing, and initial characterization of a novel ADA gene, ADA1. Similar to the previously isolated ada mutants, ada1 mutants display decreases in transcription from various reporters. Furthermore, ADA1 interacts with the other ADAs in the ADA/GCN5 complex as demonstrated by partial purification of the complex and immunoprecipitation experiments. We estimate that the complex has a molecular mass of approximately 2 MDa. Previously, it had been demonstrated that ada5 mutants displayed more severe phenotypic defects than the other ada mutants (G. A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996; S. M. Roberts and F. Winston, Mol. Cell. Biol. 16:3206-3213, 1996). ada1 mutants display defects similar to those of ada5 mutants and different from those of the other mutants with respect to promoters affected, inositol auxotrophy, and Spt ؊ phenotypes. Thus, the ADAs can be separated into two classes, suggesting that the ADA/GCN5 complex may have two separate functions. We present a speculative model on the possible roles of the ADA/GCN5 complex.RNA polymerase II (Pol II)-dependent transcription in eukaryotes requires general transcription factors and gene-specific transcriptional activators. The general factors include Pol II itself, the TATA binding protein (TBP), TFIIB, and other factors required for preinitiation complex formation at promoters. Activators bind to upstream activating sequences (UAS) or enhancers, which can be hundreds of base pairs upstream from the site of initiation of transcription, and stimulate transcription through their activation domains. In addition to these two types of factors, a third type whose members are referred to as mediators, coactivators, or adaptors has been isolated (3, 32, 44). These have been proposed to function by several mechanisms, including bridging interactions between activator proteins and basal factors and counteracting of the repressive effects on transcription by nucleosomes. Many factors believed to belong to this coactivator group form large heteromeric complexes. For example, the SWI/SNF complex is approximately 2 MDa in size (11,40,60) and functions by countering chromatin-mediated repression (14, 27). The suppressor of RNA polymerase B (SRB) complex is required for robust activated and basal transcription and interacts with basal factors to form an RNA Pol II holoenzyme complex (25,35,58). The TFIID complex consists of basal transcription factor TBP and associated TAF proteins which are thought to be required for activated transcription (15,51,59).Two other groups of factors which seem to function as adaptors or coactivators, the SPTs and the ADAs, were identified genetically in Saccharomyces cerevisiae. The SPT genes were isolated as suppressors of the auxotrophies resulting from transposon insertions at the promoters of various biosynthetic genes. The SPT genes fall into two classes based upon th...

The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors

“…However, the various subunits of the Ada complex may have distinct coactivator functions, as they are not similarly involved in activation of transcription from different promoters. For example, activation through the ADH1 and CYC1 UAS1 yeast promoters is affected in ⌬ada1 and ⌬ada5, but not in ⌬ada2, ⌬ada3, or ⌬gcn5 strains (Marcus et al 1996;Horiuchi et al 1997), whereas our present study indicates that Ada2 and Gcn5 are partially dispensable in the Ada complex recruited to the URA3 promoter through interaction between the liganded LBD of ER␣ and Ada3.…”

“…Direct and independent interactions have been observed in vitro between Ada2 and both Ada3 and Gcn5 (Horiuchi et al 1995). In addition, immunoprecipitation assays and biochemical purification of Ada complexes have revealed further association of these three proteins with Ada1 and Ada5 (Marcus et al 1996;Grant et al 1997;Horiuchi et al 1997). Here, we demonstrate that Ada2 and Gcn5, in addition to Ada3, are required for efficient ligand-dependent activation by the AF-2 of RXR␣ and ER␣, indicating that the activity of these AF-2s is most probably mediated by an Ada complex through direct interaction between the liganded LBD and the Ada3 subunit.…”

“…Ada3 is found in vivo within multisubunit protein complexes of different size (∼0.2, 0.8-0.9, and 1.8-2 MD) and complexity that contain at least three to four additional proteins: Ada1, Ada2, Gcn5 (Ada4), and Ada5 (Grant et al 1997;Horiuchi et al 1997;Saleh et al 1997). Direct and independent interactions have been observed in vitro between Ada2 and both Ada3 and Gcn5 (Horiuchi et al 1995).…”

“…Thus, Ada3 may mediate the ER␣ AF-2 activity through both Ada2/Gcn5-dependent and independent pathways. Interestingly, ada5 and ada1 mutants exhibit more severe phenotypic defects and a broader spectrum of tran- scriptionally affected genes than the ada2 and gcn5 mutants (Marcus et al 1996;Horiuchi et al 1997), suggesting that like Ada3, Ada1 and Ada5 possess additional functions that are independent of Ada2 and Gcn5 (see below). Several subunits of the Ada complexes have been shown to interact directly in vitro with the ADs of several activator proteins: Interactions have been reported between Ada2 and either VP16 (Silverman et al 1994), Gcn4 (Barlev et al 1995), Adr1 (Chiang et al 1996), or GR ( 1) (Henriksson et al 1997), between Gcn5 and Adr1 (Chiang et al 1996), and between Ada5 and VP16 (Marcus et al 1996).…”

“…SPT3 has been shown to interact genetically and functionally with TBP and TFIIA (Eisenmann et al 1992;Madison and Winston 1997). Importantly, a native 1.8-MD Ada complex (named SAGA) has been recently isolated from yeast nuclei; it contains Spt7 and Spt3, and most probably Spt8, in addition to Gcn5, Ada2, Ada3, Ada1, and Ada5 (Grant et al 1997;Horiuchi et al 1997). Moreover, a physical interaction has been demonstrated between Ada5/Spt20 and the four other Spt proteins, Spt3, Spt7, Spt8, and TBP (Grant et al 1997;Roberts and Winston 1997).…”