A functional role for the switch 2 region of yeast RNA polymerase II in transcription start site utilization and abortive initiation

RNA polymerase II (RNAPII) is responsible for the synthesis of mRNA from eukaryotic protein-encoding genes. In this study, site-directed mutagenesis was employed to probe the function of residues within the Saccharomyces cerevisiae RNAPII active center in the mechanism of transcription start site utilization. We report here the identification of two mutations in the switch 2 region, rpb1-K332A and rpb1-R344A, which conferred conditional growth properties and downstream shifts in start site utilization. Analyses of double mutant strains demonstrated functional interactions between these switch 2 mutations and a mutation in the largest subunit of transcription factor UF (TFIIF) that confers upstream shifts in start site usage. Importantly, biochemical analyses demonstrated that purified Rpb1-R344A mutant polymerase exhibited impaired ability to stabilize a short RNA-DNA hybrid in the active center, an increased frequency of abortive transcription in runoff assays, and both a downstream shift and increased abortive initiation in reconstituted transcription assays. These results provide evidence for a role of switch 2 during start site utilization and indicate that RNA-DNA hybrid stability at the 3′-end of the transcript is a determinant in this process. We discuss these results within the context of a proposed model regarding the concerted roles of RNA-PII, TFIIB, and TFIIF during mRNA 5′-end formation in S. cerevisiae.