Activation of a Poised RNAPII-Dependent Promoter Requires Both SAGA and Mediator

• Published in: Genetics (Austin) Vol. 184; no. 3; pp. 659 - 672
• Main Authors: Lee, Sarah K, Fletcher, Aaron G. L, Zhang, Lei, Chen, Xu, Fischbeck, Julie A, Stargell, Laurie A
• Format: Journal Article
• Language: English
• Published: United States Genetics Soc America 01.03.2010; Genetics Society of America
• Subjects: Binding sites; Cell growth; Cytochromes c - biosynthesis; Cytochromes c - genetics; Gene Deletion; Gene expression; Gene Expression Regulation, Fungal - physiology; Genes, Fungal - physiology; Investigations; Promoter Regions, Genetic - physiology; Proteins; RNA polymerase; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - biosynthesis; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; TATA-Box Binding Protein - genetics; TATA-Box Binding Protein - metabolism; Trans-Activators - genetics; Trans-Activators - metabolism; Transcription, Genetic - physiology
• ISSN: 0016-6731; 1943-2631; 1943-2631
• DOI: 10.1534/genetics.109.113464

A growing number of promoters have key components of the transcription machinery, such as TATA-binding protein (TBP) and RNA polymerase II (RNAPII), present at the promoter prior to activation of transcription. Thus, while transcriptional output undergoes a dramatic increase between uninduced and induced conditions, occupancy of a large portion of the transcription machinery does not. As such, activation of these poised promoters depends on rate-limiting steps after recruitment of TBP and RNAPII for regulated expression. Little is known about the transcription components required in these latter steps of transcription in vivo. To identify components with critical roles in transcription after recruitment of TBP in Saccharomyces cerevisiae, we screened for loss of gene expression activity from promoter-tethered TBP in >100 mutant strains deleted for a transcription-related gene. The assay revealed a dramatic enrichment for strains containing deletions in genes encoding subunits of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex and Mediator. Analysis of an authentic postrecruitment-regulated gene (CYC1) reveals that SAGA occupies the promoter under both uninduced and induced conditions. In contrast, Mediator is recruited only after transfer to inducing conditions and correlates with activation of the preloaded polymerase at CYC1. These studies indicate the critical functions of SAGA and Mediator in the mechanism of activation of genes with rate-limiting steps after recruitment of TBP.