UBC Theses and Dissertations
Diverse mechanisms of transcription regulation by RNA polymerase II in saccharomyces ceresisiae Aristizabal, Maria
Transcription is the process by which information encoded in the DNA is read by the cell. RNAPII is a key enzyme in this process and it contains a unique and functionally conserved C-terminal domain (CTD) composed of heptapeptide repeats. The CTD plays essential roles in the coordination of transcription events, which is is mediated by its ability to be differentially modified throughout the transcription cycle. As such, a number of CTD modifying enzymes act at different stages on the CTD, including the phosphatase Fcp1, which associates with RNAPII along the length of transcribed genes and plays key roles in transcription elongation and recycling. In addition to the modification status, both CTD sequence and length are important for normal function. This thesis focuses on understanding the role of the RNAPII-CTD and its modifying enzyme, Fcp1, in gene expression and in understanding how the CTD and the process of transcription contribute to genome instability. We found clear evidence that despite their general role in transcription both CTD length and Fcp1 function have gene-specific effects on transcription. In particular, we show that truncation of the CTD results in an increasing number of transcriptional defects. These defects resulted primarily from changes in initiation and were suppressed by loss of a previously reported CTD suppressor, CDK8. Additionally, our work on Fcp1 mutants provides a framework for exploring a general role for Fcp1 in the regulation of transcription factors. Furthermore, we find that truncating the CTD also resulted in altered expression of mobile genetic elements, a previously unreported phenotype. Here, we show that loss of CDK8 was able to suppress the increased expression of retrotransposons, expanding our understanding of retrotransposon regulation in vivo. We also find that this effect is mediated by the transcription factors Ste12 and Tec1 and that loss of either of these can suppress growth and retrotransposon expression defects of CTD truncation mutants. Finally, we report the genome-wide distribution of DNA:RNA hybrids in S. cerevisiae. By comparing this profile to that of mutants in genes encoding RNA or DNA:RNA hybrid processing factors, we show differential effects on DNA:RNA hybrid abundance and localization.
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