RNA polymerase II-TFIIE-TFIIH interface functions in transcription start site selection in Saccharomyces cerevisiae

During transcription initiation in Saccharomyces cerevisiae , RNA polymerase II (Pol II) and the general transcription factors (GTFs) assemble upstream of transcription start sites (TSSs) to form the pre-initiation complex (PIC). In this model organism, yeast, the PIC selects TSSs through a unidirectional scanning mechanism referred to as promoter scanning. Previous studies have shown that the TFIIH subunit Tfb3 connects TFIIH to the rest of the PIC via interactions with (i) Pol II, (ii) TFIIE, and iii) other TFIIH components. Activities within the PIC that influence TSS selection can do so by control of initiation efficiency at individual TSSs and by control of TSS scanning (either rate of scanning or scanning processivity). Here, we identified classes of tfb3 and tfa1 mutants within the TFIIH-Pol II-TFIIE interface able to alter promoter scanning in either upstream or downstream directions, suggesting that changes to this interface can fine-tune scanning behavior through evolution. Mutants were isolated based on transcriptional phenotypes and were validated for changes to promoter-specific TSS usage at ADH1 . Subsets of alleles were analyzed using TSS sequencing approaches, showing that tested tfb3 and tfa1 alleles shift TSS distributions across most genomic promoters. Genetic interaction and genomic analysis revealed that the Tfb3 interfaces with Rpb7 and Tfa1 both contribute to promoter scanning, and that tfb3 alleles exhibit additive effects with ssl2 and sub1 Δ, consistent with Tfb3-PIC interactions likely modulating scanning processivity.