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Nat Struct Mol Biol. 2008 Aug;15(8):811-8. doi: 10.1038/nsmb.1458. Epub 2008 Jun 13.

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

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Gene Center and Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universit√§t M√ľnchen, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.


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 alpha-amanitin locked the elongation complex in a new state, which was refined at 3.4-A 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 A 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 alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix.

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