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Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation

Abstract

To study how RNA polymerase II translocates after nucleotide incorporation, we prepared elongation complex crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor α-amanitin locked the elongation complex in a new state, which was refined at 3.4-Å resolution and identified as a possible translocation intermediate. The DNA base entering the active site occupies a 'pretemplating' position above the central bridge helix, which is shifted and occludes the templating position. A leucine residue in the trigger loop forms a wedge at the shifted bridge helix, but moves by 13 Å to close the active site during nucleotide incorporation. Our results support a Brownian ratchet mechanism that involves swinging of the trigger loop between open, wedged and closed positions, and suggest that α-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix.

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Figure 1: Structure of the α-amanitin–inhibited Pol II EC.
Figure 2: Contacts between α-amanitin and Pol II.
Figure 3: The α-amanitin–inhibited EC structure is apparently a translocation intermediate.
Figure 4: Comparisons with bacterial RNA polymerase and possible EC states.
Figure 5: Model of the nucleotide addition cycle (NAC).

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Acknowledgements

We thank D. Kostrewa and other members of the Cramer laboratory for help. We thank D. Temiakov for discussions. F.B. was supported by the Nanosystems Initiative Munich (NIM) and the Elitenetzwerk Bayern (ENB). P.C. was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich SFB646, the Transregio 5 Chromatin, the EU research grant network 3D Repertoire and the Fonds der Chemischen Industrie. Part of this work was performed at the Swiss Light Source (SLS) at the Paul Scherrer Institut, Villigen, Switzerland.

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Correspondence to Patrick Cramer.

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Brueckner, F., Cramer, P. Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation. Nat Struct Mol Biol 15, 811–818 (2008). https://doi.org/10.1038/nsmb.1458

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