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Nguyen, Thomas Fang

University of British Columbia

Transcription in eukaryotes is primarily performed by three distinct types of RNA polymerases: RNA polymerases I, II, and III (Pol I, Pol II, and Pol III). Decades of research have revealed a comprehensive understanding of the mechanisms of eukaryotic transcription; however, advances in technologies may require a reassessment of previous studies, potentially unveiling previously unattainable insights. Consequently, my thesis focuses on using cutting edge technologies such as super resolution microscopy, next-generation sequencing, and mass spectrometry-based proteomics to delve into the intricate mechanisms of Pol II and Pol III transcription. TATA-box binding protein (TBP) was previously known to be indispensable by all three polymerases for transcription initiation in most eukaryotes. Here, I present evidence for a TBP-family independent mechanism of Pol II transcription in mouse embryonic stem cells, showing that ongoing transcription, gene induction, and TFIID formation are not affected when TBP is depleted, while Pol III transcription is severely impacted. In addition, I demonstrate that heat shock stress leads to a drastic change in the transcription of tRNA genes by Pol III, influenced by the heat shock master regulator HSF1. Lastly, I use super resolution microscopy to explore the dynamics of the general transcription factors through single-particle tracking, demonstrating significant changes in residence time and DNA binding when TBP is depleted. Taken together, my thesis aims to unravel novel perspectives of transcription mechanisms using new technologies, but also to challenge previous paradigms and provide a more comprehensive understanding of gene regulation.

Text

2024-04-26

Attribution 4.0 International

http://hdl.handle.net/2429/88026

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by/4.0/