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 (gcn5Δ spt3Δand gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Δ, spt20Δ, or ada1Δmutants. 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.

ACKNOWLEDGMENTS

S. M. Roberts and P. A. Grant contributed equally to this work.

We thank R. Candau for preparation of the ADA1 disruption strain SB11, Z. Yang for technical assistance in the preparation of Spt7ΔBrD HAT complexes, and N. Barlev for advice on binding assays. We thank R. Reeder for the gift of GST-TBP plasmid, J. Reese and M. Green for Taf_II antibodies, and A. Navas, Z. Zhou, and S. Elledge for informing us that spt20 mutants have an HU^S phenotype. We thank G. Moore for helpful discussions and critical comments on the manuscript.

This research was supported by grants from the National Institutes of General Medical Sciences to S.L.B., F.W., and J.L.W. and from the National Science Foundation and the Council for Tobacco Research to S.L.B. P.A.G. was supported by a postdoctoral fellowship from the American Cancer Society. An NIH Cancer Core training grant to the Wistar Institute supported D.E.S. and L.J.D.; D.E.S. was also supported by an NIH postdoctoral fellowship.