Inhibition of TATA-binding protein function by SAGA subunits Spt3 and Spt8at Gcn4-activated promoters

SAGA is a 1.8-MDa yeast protein complex that is composed of several distinc t classes of transcription-related factors, including the adaptor/acetyltra nsferase Gcn5, Spt proteins, and a subset of TBP-associated factors. Our re sults indicate that mutations that completely disrupt SAGA (deletions of SP T7 or SPT20) strongly reduce transcriptional activation at the HIS3 and TRP 3 genes and that Gcn5 is required for normal HIS3 transcriptional start sit e selection. Surprisingly, mutations in Spt proteins involved in the SAGA-T BP interaction (Spt3 and Spt8) cause derepression of HIS3 and TRP3 transcri ption 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 induce HIS3 and T RP3 transcription result in an altered balance between these complexes stro ngly in favor of the form without Spt8, These results suggest that the comp osition of SAGA may be dynamic in vivo and may be regulated through dissoci able inhibitory subunits.