Single molecule studies of RNA polymerase II transcription initiation and elongation
CitationBaek, Inwha. 2021. Single molecule studies of RNA polymerase II transcription initiation and elongation. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.
AbstractIn eukaryotes, RNA polymerase II (Pol II) synthesizes messenger RNAs and small non-coding RNAs. For accurate and efficient transcription, Pol II works with numerous factors, forming stage-specific Pol II complexes. There has been remarkable progress in our understanding of these Pol II complexes during the past decades. However, dynamic aspects of Pol II complex formation remain poorly characterized. In this dissertation, I present the dynamics of factor addition during activator-dependent preinitiation complex (PIC) and elongation complex assembly and provide a quantitative kinetic model for activator-dependent Pol II transcription.
I implemented multi-wavelength single molecule fluorescence microscopy on nuclear extract from Saccharomyces cerevisiae. Yeast nuclear extract has been shown to faithfully recapitulate activator-dependent PIC assembly, initiation and elongation, as it contains the full repertoire of nuclear proteins. Single molecule imaging revealed branched pathways for activator-dependent PIC assembly where Pol II and Pol II-associated factors TFIIF and TFIIE are initially recruited to enhancer-bound activators before loading into a PIC at the promoter. Furthermore, multiple molecules of Pol II, TFIIF and TFIIE simultaneously bound to a single enhancer, suggesting that they create an activator-mediated localized cluster at enhancers. Unlike Pol II, TFIIF and TFIIE, TFIIH binding was singular and dependent on the core promoter. Additionally, unexpected dynamics of factors such as multiple cycles of TFIIE binding and release were observed.
Single molecule measurements also revealed that a slow step needs to occur after initial Pol II recruitment to enhancer to form a PIC and elongation complex at the core promoter. This slow step, predicted to be a major rate-limiting step for PIC and elongation complex assembly, manifested in a lag before TFIIH binding to DNA. The elongation factor Spt4/5 also exhibited a lag before binding to DNA, reflecting step(s) required for PIC assembly and the transition from initiation to elongation. Through single molecule imaging in the highly complex environment of nuclear extract, this work uncovers the branched pathways for activator-dependent PIC assembly and provides a comprehensive quantitative kinetic model for PIC and elongation complex formation. Moreover, this work establishes a fundamental framework for probing the dynamics of the transcription process.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37368331
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