Inhibition of TATA-Binding Protein Function by SAGA Subunits Spt3 and Spt8 at Gcn4-Activated Promoters

Abstract

SAGA is a 1.8-MDa yeast protein complex that is composed of several distinct classes of transcription-related factors, including the adaptor/acetyltransferase Gcn5, Spt proteins, and a subset of TBP-associated factors. Our results indicate that mutations that completely disrupt SAGA (deletions of SPT7 orSPT20) strongly reduce transcriptional activation at the HIS3 and TRP3 genes and that Gcn5 is required for normal HIS3 transcriptional start site selection. Surprisingly, mutations in Spt proteins involved in the SAGA-TBP interaction (Spt3 and Spt8) cause derepression of HIS3 andTRP3 transcription in the uninduced state. Consistent with this finding, wild-type SAGA inhibits TBP binding to the HIS3 promoter in vitro, while SAGA lacking Spt3 or Spt8 is not inhibitory. We detected two distinct forms of SAGA in cell extracts and, strikingly, one lacks Spt8. Conditions that induceHIS3 and TRP3 transcription result in an altered balance between these complexes strongly in favor of the form without Spt8. These results suggest that the composition of SAGA may be dynamic in vivo and may be regulated through dissociable inhibitory subunits.

ACKNOWLEDGMENTS

We thank F. Winston and L. Pacella heartily for valuable discussions and for generosity in providing the many SPT strains, plasmids, and antibodies used in this study. We also thank J. Workman, P. Grant, and members of the Workman laboratory as well as Thanos Halazonetis for advice and help in the fractionation of SAGA and SAGA_alt. We thank J. Reese and M. Green for TAF_II antibodies and A. Hinnebusch for Gcn4 antibodies and the kind gift of plasmids for GCN4 gene deletion and constitutive Gcn4 expression. We thank G. Moore, J. Workman, and members of the Berger laboratory for helpful discussions and critical comments on the manuscript.

D.E.S. was supported by a postdoctoral fellowship from the National Institutes of Health and by an NIH Cancer Core training grant to The Wistar Institute. This research was supported by grants from the National Institute of General Medical Sciences and the National Science Foundation to S.L.B. and the National Institute of General Medical Sciences to P.M.L.