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978-3-8439-0591-6, Reihe Biophysik

Barbara Treutlein
Mechanisms of eukaryotic gene expression on a single molecule level: From transcription initiation to nucleosome remodeling

226 Seiten, Dissertation Ludwig-Maximilians-Universität München (2012), Softcover, B5

Zusammenfassung / Abstract

This thesis focuses on understanding the molecular mechanisms of two fundamental processes of eukaryotic gene expression: transcription initiation and nucleosome remodeling. Since both processes are characterized by large conformational changes and a high flexibility of nucleoprotein complexes, standard high-resolution structural methods are hindered and instead direct visualization in real time is required as provided by single molecule techniques.

In the first part of this work, I used single molecule fluorescence resonance energy transfer (smFRET) experiments, Nano-Positioning System (NPS) analysis and x-ray crystallographic information to determine the three-dimensional architecture of a minimal Pol II open promoter complex (OC) consisting of promoter DNA, TBP, Pol II and general transcription factors TFIIB and TFIIF. In the OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. Furthermore, I directly observed the downstream DNA to be dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. These results uncover large overall structural changes during the initiation-elongation transition.

In the second part, I applied the same experimental approach to determine the location of the three constituting domains of chromatin remodeler Chd1 (Chromodomain-helicase-DNA-binding protein 1) in a Chd1-nucleosome complex that exhibited intrinsic dynamics. The NPS results allowed me to construct a model of the Chd1-nucleosome complex, in which the DNA-binding domain is associated with extranucleosomal DNA at the nucleosome entry site, the tandem chromodomains are located below the entry site close to histone H4 and H3 tails and the ATPase motor binds nucleosomal DNA between dyad and superhelical location +1. Furthermore, I used smFRET to follow the structural dynamics of nucleosomal DNA during Chd1 catalyzed repositioning in real-time. FRET time trajectories revealed gradual and bidirectional translocation of nucleosomal DNA by Chd1 and the data allowed me to propose a model for the remodeling mechanism of Chd1, which involves formation and propagation of a DNA loop.