Genetic dissection of transcription start sites' selection by RNA Pol II in Saccharomyces cerevisiae

Transcription initiation by RNA Pol II in Saccharomyces cerevisiae involves the participation of dozens of proteins including the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH that together with Pol II form the preinitiation complex. A subset of these proteins have been implicated to participate in transcription start sites selection wherein mutations result in shift of transcription start sites (TSSs). However, most of the pre-initiation complex is not studied in the context of TSS shifts. To ask what factors or residues participate in TSS selection such that mutations in them result in TSS shifts and how they functionally participate in the process of TSS in Saccharomyces cerevisiae, we performed a genetic selection of initiation mutants as suppressors of initiation reporters. About 2000 initiation mutants were isolated as suppressors of four TSS-shift-sensitive reporters (two upstream-shift sensitive and two downstream-shift sensitive), screened by phenotype to study mutation defects and independence amongst the suppressors. Causal mutations were identified in prioritized ~300 mutants by WGS and Illumina AmpliSeq and finally 8 novel mutants expressing downstream reporters were tested for universal TSS shifts by STRIPE-seq. Mutants displayed two distinct classes of TSS shifts which related to the functional role of proteins: chromatin factors and initiation factors.