Structure-function studies of the Tfg2 subunit of Saccharomyces cerevisiae transcription factor IIF
Brodie, Seth A
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Regulation of gene expression in eukaryotic cells confers much of the complexity needed by an organism to adapt to various environments, cellular stresses and energy sources. Multi-cellular and highly complex organisms rely on the ability to alter gene expression to develop different cell types and tissues. Even more simple organisms, such as the yeast Saccharomyces cerevisiae , rely on the ability to turn genes on and off to respond to their surroundings. Although there are multiple processes to regulate gene expression, most often this is achieved at the level of RNA synthesis, i.e. transcription. TFIIF has been implicated in multiple stages of the transcription cycle. As a central component of the preinitiation complex (PIC), TFIIF is thought to be responsible for aiding in the recruitment of RNAPII to the promoter through interactions between TFIIB and polymerase, and is required for the association of TFIIE and TFIIH. TFIIF has also been shown to interact with, and stimulate the activity of, an RNAPII C-terminal domain (CTD) phosphatase designated Fcp1. This function of TFIIF is proposed to regulate the later phases of transcript elongation. Lastly, TFIIF can enhance elongation efficiency by interacting with RNAPII and suppressing pausing along the DNA template. The broad objective of this dissertation is to better understand the functions of TFIIF in the yeast S. cerevisiae transcription cycle. More specifically, the studies are focused on the function of the Tfg2 subunit of TFIIF. Presented in the following chapters are the results from mutational, biochemical, and functional interaction studies to investigate the role(s) of Tfg2 during both the initiation and elongation phases of the RNAPII transcription cycle. The major conclusions drawn from the results of these studies are (i) that Tfg2 plays a role in the mechanism of transcription start site utilization in S. cerevisiae ; (ii) that an altered TFIIF-RNAPII interaction underlies the alteration in start site utilization conferred by mutants of Tfg2; and (iii) that mutations in Tfg2 functionally interact with mutations in TFIIS, implicating a role for the Tfg2 subunit of TFIIF in transcript elongation.