Structure-function analysis of the Saccharomyces cerevisiae RNA polymerase II active center: A functional role for the switch 2 region in transcription start site utilization and abortive initiation
Majovski, Robert C.
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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. 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, is reported in this dissertation. Analyses of double mutant strains demonstrated functional interactions between these switch 2 mutations, mutations in transcription factor IIB (TFIIB), and a mutation in the largest subunit of transcription factor IIF (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. These results are discussed within the context of a proposed model regarding the concerted roles of RNAPII, TFIIB, and TFIIF during mRNA 5'-end formation in S. cerevisiae.