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

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Abstract
The crystal structure of the yeast RNA Pol II elongation complex bound to the inhibitor α-amanitin is solved, revealing that two functional elements, the trigger loop and the bridge helix, are trapped in a position different from their pre- and post-translocation states. This is proposed to be a translocation intermediate, lending support to a Brownian ratchet mechanism for RNA Pol II translocation during elongation. 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.