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Plaschka, Clemens (2015): Integrative structural and functional studies of Mediator and RNA polymerase II transcription initiation assemblies. Dissertation, LMU München: Faculty of Chemistry and Pharmacy



Gene transcription is a fundamental process of the living cell. Eukaryotic transcription of messenger RNA requires the regulated recruitment of the conserved transcribing enzyme RNA polymerase (Pol) II to the gene promoter. The most heavily regulated step is transcription initiation that involves the ordered assembly of Pol II, the general transcription factors (TF) -IIA, -IIB, -IID, -IIF, -IIE, -IIH and the co-activator Mediator complex. Mediator communicates between transcription regulators and Pol II, and is associated with human disease. Mediator from the yeast Saccharomyces cerevisiae (Sc) has a molecular mass of 1.4 megadaltons and contains 25-subunits that constitute a head, middle, tail and kinase module. The core of Mediator contains the head and middle modules that are essential for viability in Sc, and directly contact Pol II. Mediator co-operates with TFIIH, to assist assembly and stabilization of the transcription initiation complex and stimulate TFIIH kinase activity. Because of the large size and complexity of Mediator and the initiation machinery, the underlying mechanism remains poorly understood. In this work we studied the structure and function of Mediator head and middle modules, the structure of the reconstituted Pol II-core Mediator transcription initiation complex, and reveal mechanisms of transcription regulation. We report the crystal structure of the 6-subunit Schizosaccharomyces pombe Mediator head module at 3.4 Å resolution. The structure resembles the head of a crocodile and reveals eight elements that are part of three domains named neck, fixed jaw and movable jaw. The neck contains a spine, shoulder, arm and finger. The arm and essential shoulder elements contact the remainder of Mediator and Pol II. The head module jaws and central joint, important for transcription, also interact with Mediator and Pol II. The Sp head module structure is conserved and revises a 4.3 Å model of the Sc head module, explains known mutations, and provides an atomic model for one half of core Mediator. We further propose a model of the Mediator middle module based on protein crosslinking and mass spectrometry. To determine how Mediator regulates initiation, we prepared recombinant Sc core Mediator by co-expression of its 15 subunits in bacteria. Core Mediator is active in transcription assays and bound an in vitro reconstituted core initially transcribing complex (cITC) that contains Pol II, the general factors TFIIB, TBP, TFIIF, and promoter DNA. We determined the cryo-electron microscopy structure of the initially transcribing core initiation complex at 7.8 Å resolution. The structure reveals the arrangement of DNA, TBP, TFIIB, and TFIIF on the Pol II surface, the path of the complete DNA template strand and three TFIIF elements. The ‘charged helix’ and ‘arm’ of TFIIF subunit Tfg1, reach into the Pol II cleft and may stabilize open DNA. The linker region of TFIIF subunit Tfg2 extends between Pol II protrusion and TFIIB, and may stabilize TFIIB. The structure agrees with its human counterpart, and suggests a conserved architecture of the core initiation complex. Finally, we determined the cryo-electron microscopy architecture of the cITC-core Mediator complex at 9.7 Å resolution. Core Mediator binds Pol II at the Rpb4/Rbp7 stalk close to the carboxy-terminal domain (CTD). The Mediator head module contacts the Pol II dock and TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module ‘plank’ domain touches the Pol II foot and may control polymerase conformation allosterically. The Med14 subunit bridges head and middle modules with a ‘beam’, and connects to the tail module that binds transcription activators located on upstream DNA. The ‘arm’ and ‘hook’ domains of core Mediator form part of a ‘cradle’ that may position CTD and the TFIIH kinase to stimulate Pol II phosphorylation. Taken together, our results provide a structural framework to unravel the role of Mediator in transcription initiation and determine mechanisms of gene regulation.