Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

Abstract

SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5⌬ spt3⌬ and gcn5⌬ spt8⌬) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7⌬, spt20⌬, or ada1⌬ mutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.

Actively Transcribed GAL Genes Can Be Physically Linked to the Nuclear Pore by the SAGA Chromatin Modifying Complex

“…Standard deviations in the data are indicated by the error bars. not required to link actively transcribed genes to the nuclear pore, which is consistent with previous reports that Gcn5 is not required for expression of the GAL genes (23,24), integrity of the SAGA complex (43), or visual detection of the GAL genes at the nuclear periphery (9).…”

“…Deletion of GCN5 results in a catalytically inactive yet structurally intact SAGA complex (37,43). To determine whether histone acetyltransferase activity was important to link the SAGA-dependent GAL loci to the nuclear pore, we tested whether deletion of GCN5 decreases the interaction of Mlp1 with the GAL1/10 locus using ChIP.…”

The subcellular localization of the ChoRE-binding protein, encoded by the Williams–Beuren syndrome critical region gene 14, is regulated by 14-3-3

“…NIF3L1 is the ortholog of the yeast Ngg1-interacting factor 3 (NIF3) (26). The yeast yNIF3p was shown to interact with yNGG1p (52), now designated yADA3p, a subunit of histone acetyltransferase (HAT) complexes (53,54). Reciprocally the human homolog of yADA3p, ADA3, was identified in the PCAF and the hGCN5-containing HAT complexes (55).…”

Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA

“…The SAGA complex is a cofactor required for normal expression of some genes in yeast (Sterner et al 1999;Lee et al 2000). On gene activation in vivo, SAGA can be recruited to promoters independently of the general transcription factors (Bhaumik and Green 2001;Larschan and Winston 2001).…”

“…On gene activation in vivo, SAGA can be recruited to promoters independently of the general transcription factors (Bhaumik and Green 2001;Larschan and Winston 2001). The SAGA complex contains a core comprising Ada and Spt subunits, a subset of Tafs, acetyltransferase and ubiquitin protease activities, the essential factor Tra1, and two factors related to TBP function, Spt3 and Spt8 (Grant et al 1998;Sterner et al 1999;Pray-Grant et al 2002;Sanders et al 2002). SAGA is closely related to the SLIK/SALSA complex, which lacks Spt8 but which contains at least one unique subunit, Rtg2 (Pray- Grant et al 2002;Sterner et al 2002;Wu and Winston 2002).…”