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Brückner, Florian (2008): Molecular basis of translocation, alpha-amanitin inhibition, and CPD damage recognition by RNA polymerase II. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



RNA polymerase II (Pol II) is the eukaryotic enzyme responsible for transcribing all protein-coding genes into messenger RNA (mRNA). This thesis describes studies on the molecular mechanisms of Pol II translocation, alpha-amanitin inhibition and DNA lesion recognition by Pol II. To study how Pol II translocates after nucleotide incorporation, we prepared elongation complex (EC) crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the EC in a new state that we identified as a translocation intermediate at 3.4 Å resolution. The DNA base entering the active site occupies a “pre-templating” position above the central bridge helix, which is shifted and occludes the standard templating position. A leucine residue in the trigger loop forms a wedge next to the shifted bridge helix, but moves by 13 Å to close the active site for nucleotide incorporation. Our results support a Brownian ratchet mechanism of elongation that involves swinging of the trigger loop between open, wedged, and closed positions, and suggest that alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix in a translocation intermediate. Cells use transcription-coupled repair (TCR) to efficiently eliminate DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs). Here we present the structure-based mechanism for the first step in eukaryotic TCR, CPD-induced stalling of Pol II. A CPD in the transcribed strand slowly passes a translocation barrier, and enters the polymerase active site. The CPD 5’-thymine then directs uridine monophosphate (UMP) misincorporation into mRNA, which blocks translocation. Artificial replacement of the UMP by adenosine monophosphate (AMP) enables CPD bypass, thus Pol II stalling requires CPD-directed misincorporation. In the stalled complex, the lesion is inaccessible, and the polymerase conformation is unchanged. This is consistent with non-allosteric recruitment of repair factors and excision of a lesion-containing DNA fragment in the presence of Pol II. CPD recognition is compared with the recognition of a cisplatin-induced guanine-guanine intrastrand crosslink. Similarities and differences in the detailed mechanism of transcriptional stalling at the two different dinucleotide lesions are discussed.