Previous studies have shown that substitutions in the Tfg1 or Tfg2 subunits of S. cerevisiae transcription factor IIF (TFIIF) can cause upstream shifts in start site utilization, resulting in initiation patterns that more closely resemble those of higher eukaryotes. In this study, we report the results from multiple biochemical assays analyzing the activities of wild-type and the Tfg1-E346A mutant yeast TFIIF. We demonstrate that TFIIF stimulates formation of the first two phosphodiester bonds, dramatically stabilizes a short RNA*DNA hybrid in the RNA polymerase II (RNAPII) active center, and importantly, that the Tfg1-E346A substitution coordinately enhances early bond formation and the processivity of early elongation in vitro. These results are discussed within a proposed model for the role of yeast TFIIF in modulating conformational changes in the RNAPII active center during initiation and early elongation.
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