Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations (RNA)

Three general classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA and Mediator (MED) Tail, but little is known about whether this dependence is determined by the core promoter, Upstream activation sites (UAS), or other gene features. It is also unclear whether UASs can broadly activate transcription from the different promoter classes or whether efficient transcription requires matching UASs and promoters of similar gene class. Here we measure transcription and cofactor specificity for tens of thousands of UAS-core promoter combinations. We find that few UASs display strong core promoter specificity while most UASs can broadly activate promoters regardless of regulatory class. However, we find that matching UASs and promoters from the same gene class is generally important for optimal expression. We find that MED Tail and SAGA are dependent on the identity of both UAS and promoter while dependence on TFIID localizes to only the core promoter. Overall design: Tens of thousands of UAS-core promoter combinations were cloned into a barcoded transcriptional reporter. Transcription was measured using short 4-TU labeling and MTS-biotin enrichment +/- acute auxin-mediated depletion of Taf13 (TFIID), Spt7 (SAGA) or Med15 (Mediator) subunits. Reporter transcripts were matched to UAS-core combinations by sequencing reporter plasmid library, which was also used to normalize transcription values.