Mechanisms of yeast RNA polymerase II transcription and the role of transcription factor IIF
MetadataShow full item record
Transcription of protein-coding genes by eukaryotic RNA polymerase II (RNAPII) is a multistep process that involves the concerted action of RNAPII and accessory proteins including the general transcription factors (GTFs); TFIID, TFIIB, TFIIF, TFIIE and TFIIH. The GTFs are being intensely studied to determine their function during the different stages of the transcription cycle. Numerous investigations have reported functions for the general transcription factor IIF (TFIIF) of higher eukaryotes in multiple stages of the transcription cycle, although few studies have examined the TFIIF homolog in the yeast Saccharomyces cerevisiae. The objective of this dissertation is to better understand the mechanism of action of S. cerevisiae TFIIF during the different stages of the RNAPII transcription cycle. Although the basal transcription factors are highly homologous in eukaryotes, the mechanism of transcription start site utilization on TATA-dependent promoters in S. cerevisiae is fundamentally different from that in higher eukaryotes, where the PIC assembles on the promoter and transcription initiates at a discrete site 25-30 base pairs downstream of the TATA element with the architecture of the PIC determining the initiation site. In contrast, the S. cerevisiae RNAPII machinery typically initiates at multiple sites in a window from 45 to 120 base pairs downstream of the TATA element. Results in this thesis support a transcription initiation mechanism that involves transcription-independent translocation of the yeast RNAPII to the far downstream start sites. Previous work in our laboratory identified mutations in the yeast TFIIF subunits Tfg1 and Tfg2 that confer upstream shifts in start site utilization. In vivo and in vitro studies demonstrate that TFIIF modulates the utilization of transcription start site sequences through its interaction with RNAPII. In the second and third part of this dissertation, genetic and biochemical approaches were utilized to better define TFIIF functions at post-initiation steps in the S. cerevisiae transcription cycle, and support a role for TFIIF in both promoter escape and early elongation. Combined results of this work support a model for TFIIF function where the TFIIF, through its interaction with RNAPII, affects the DNA recognition properties of the polymerase during start site utilization, promoter escape, and early elongation.