Structural and functional studies of RNA polymerase II passage through nucleosomes
Dissertation
Datum der mündl. Prüfung:2024-02-02
Erschienen:2024-03-05
Betreuer:Prof. Dr. Patrick Cramer
Gutachter:Prof. Dr. Patrick Cramer
Gutachter:Prof. Dr. Rubén Fernández-Busnadiego
Dateien
Name:Ochmann_DoctoralThesis.pdf
Size:187.Mb
Format:PDF
Description:Accepted doctoral thesis by Moritz Ochmann
Diese Datei ist bis 01.02.2025 gesperrt.
Zusammenfassung
Englisch
Transcription by RNA polymerase II (Pol II) is an essential process of life occurring in the context of the compact eukaryotic nucleus. While transcription elongation has been extensively studied on linear DNA in vitro, our understanding of transcription on physiologically relevant chromatinized templates, consisting of long arrays of nucleosomes, remains severely limited. Recently, several structures provided snapshots of the yeast RNA polymerase II transcribed into a single nucleosome. However, information regarding the mechanism, the required components and the dynamics of nucleosome transcription are lacking. In this thesis, I extend our limited knowledge of transcription elongation by providing extensive biochemical and structural characterization of the process of promoter-proximal pausing as well as productive elongation in the context of nucleosomes. In order to dissect the role the +1 nucleosome plays during promoter-proximal pausing, the kinetic bottleneck of transcription that occurs in proximity to the nucleosome under physiological conditions, I established a novel in vitro transcription system. Here, I show that, in the presence of pausing factors DSIF and NELF, a nucleosome causes promoter-proximal pausing at the precise position observed in vivo. Additionally, I present data to show that a weakened nucleosomal entry barrier, in the form of a hexasome, results in reduced pausing. Furthermore, I report that the histone chaperone FACT does not modulate the entry barrier and thus does not affect pausing. In stark contrast to the efficient productive elongation of chromatin in vivo, Pol II struggles to overcome a single nucleosome in current in vitro transcription systems. In this study, I dissect the effect that elongation factors of the activated elongation complex (EC*) have on nucleosome transcription under physiological conditions. I report that RTF1 has the strongest stimulatory effect, leading to three times more Pol II overcoming the nucleosome barrier. Additionally, I confirm that nucleosome transcription is accompanied by backtracking and that TFIIS is essential to recover and restart backtracked Pol II, showing TFIIS’ integral role during chromatin transcription. Furthermore, we report the first structures of a backtracked EC* with a downstream hexasome as well as the EC* and EC bound by TFIIS. These structures show new interactions between the transcription machinery and the downstream nucleosome, as well as the binding of TFIIS to mammalian Pol II. While elongation factors improve Pol II’s ability to transcribe and thereby displace nucleosomes, the histone chaperone FACT not only facilitates chromatin transcription but also minimizes chromatin disruption by Pol II. I present biochemical data showing how FACT stimulates transcription elongation, by reducing the strong dyad barrier, on both a single nucleosome and nucleosome array. Additionally, we present the first mammalian structure of the EC* with an upstream FACT-bound hexasome. The structure reveals how FACT stabilizes the barely rewrapped hexasome and shows the first structural link between FACT and the transcription machinery. The observed interaction between RTF1’s FACT fastener helix and SPT16 may provide the first evidence of the molecular basis for elongation factor mediated histone transfer around Pol II. Taken together, these results greatly advance our understanding of the dynamic processes that occur during chromatin transcription and potentially provide the first indication of the elusive histone transfer mechanism. Furthermore, by significantly improving the efficiency of in vitro nucleosome transcription under physiological conditions, this work provides the foundation for future studies that continue to decipher the mechanisms behind chromatin transcription in more detail, as well as investigating co-transcriptional chromatin processes.
Keywords: Transcription; Chromatin; Elongation; RNA polymerase II; Pausing; Promoter-proximal pausing; Productive elongation; Nucleosome; Nucleosome passage