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Kettenberger, Hubert (2005): Structure of the Complete RNA Polymerase II Elongation Complex and its Interaction with the Elongation Factor TFIIS. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



This thesis describes crystal structures of complete, 12-subunit yeast RNA polymerase II (Pol II) in complex with a synthetic transcription bubble and product RNA, with an NTP substrate analogue, and in complex with the transcription elongation factor TFIIS. The structure of the Pol II-transcription bubble-RNA complex reveals incoming template and non-template DNA, a seven base-pair DNA-RNA hybrid, and three nucleotides each of separating DNA and RNA. Based on this structure, those parts of Pol II were identified which are involved in separating template DNA from non-template DNA before the active site, and DNA from product RNA at the upstream end of the DNA-RNA hybrid. In both instances, strand separation can be explained by Pol II-induced duplex distortions. Only parts of the complete transcription bubble present in the complexes are ordered in the crystal structure, explaining the way in which high processivity of Pol II is reconciled with rapid translocation along the DNA template. The presence of an NTP substrate analogue in a conserved putative pre-insertion site was unveiled in a Pol II-transcription bubble-RNA complex crystal soaked with the substrate analogue GMPCPP. The structure of the Pol II-TFIIS complex was obtained from Pol II crystals soaked with TFIIS. TFIIS extends from the Pol II surface to the active site and complements the active site with two essential and invariant acidic residues for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that reposition nucleic acids, in particular RNA, near the active centre. These results support the idea that Pol II contains a single tuneable active site for RNA polymerisation and cleavage. The technical obstacles imposed by crystal structure determination of large, transient protein-DNA-RNA complexes were overcome by two novel, fluorescence-based assays to monitor and optimise the composition of the crystals. Both assays are not limited to Pol II complexes, but can serve as a general tool for the crystallographic community.