Functions of Saccharomyces cerevisiae TFIIF during Transcription Start Site Utilization

Abstract

Previous studies have shown that substitutions in the Tfg1 or Tfg2 subunits of Saccharomyces 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 yeast TFIIF and the TFIIF Tfg1 mutant containing the E346A substitution (Tfg1-E346A). We demonstrate that TFIIF stimulates formation of the first two phosphodiester bonds and 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.

ACKNOWLEDGMENTS

We thank Nancy Woychik for yeast strain WY9, Caroline Kane for plasmid pET15b/IIS 1-309, and Steve Hahn for communicating results prior to publication. We also thank Jim Hernandez for technical advice about the analysis of short RNAs and members of the Ponticelli laboratory for helpful discussions and comments on the manuscript.

This research was supported by National Institutes of Health Public Health service grant GM51124 (to A.S.P.).