Functions of Saccharomyces cerevisiae TFIIF during transcription start site utilization.
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.
Khaperskyy
Denys A
DA
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14214-3000, USA.
Ammerman
Michelle L
ML
Majovski
Robert C
RC
Ponticelli
Alfred S
AS
D019943:Amino Acid Substitution; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D029701:Saccharomyces cerevisiae Proteins; D019473:Transcription Factors, TFII; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II; C056604:transcription factor TFIIF