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RNA polymerase II holoenzyme-mediated regulation of GAL4 Hirst, John Martin


Much of the current understanding of eukaryotic transcriptional regulation is based on insights gained through the use of model systems. The premier transcriptional model system is galactose metabolism in the yeast Saccharomyces cerevisiae, regulated by the transcriptional activator GAL4. Regulation by GAL4 involves a number of mechanisms that act to ensure that galactose is catabolized when available, but that glucose is used preferentially. One such mechanism involves phosphorylation of GAL4 at serine 699. Phosphorylation at S699 is required for efficient galactose-inducible transcription of the GAL genes, but occurs as a consequence of transcriptional activation by GAL4. This has led to the hypothesis that phosphorylation occurs through interaction with RNA polymerase II holoenzyme components. The experiments described in this thesis determine the role of RNA polymerase II holoenzyme-associated kinases in phosphorylation of GAL4, and investigate the mechanism through which phosphorylation at S699 affects induction. Data presented here demonstrate that GAL4 S699 is a substrate for the cyclin dependent protein kinase SRBTO. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 in vitro. RNA polymerase II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo, whereas holoenzymes from srblO yeast are incapable of phosphorylating GAL4 at S699. SRB10 is required for efficient GAL induction and is shown to be epistatic to S699 phosphorylation. To investigate interactions between GAL4 and it's regulator GAL80, a two-hybrid based strategy was devised and used to demonstrate that S699 phosphorylation may regulate interaction between GAL4 and GAL80 in vivo. These data define a function for the SRB10 holoenzyme-associated CDK8 that involves regulation of transactivators by phosphorylation during transcriptional activation.

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