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Functions of Saccharomyces cerevisiae TFIIF during Transcription Start Site Utilization

Denys A. Khaperskyy, Michelle L. Ammerman,^, Robert C. Majovski, and Alfred S. Ponticelli^*

Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214-3000

Received 21 December 2007/ Returned for modification 5 February 2008/ Accepted 13 March 2008

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.

Published ahead of print on 24 March 2008.

These authors contributed equally to this work.

Current address: Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14214.

Current address: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 11724.