Structural basis of transcription inhibition by alpha-amanitin and implications for RNA polymerase II translocation.
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.
Gene Center and Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Florian
F
Brueckner
Patrick
P
Cramer
D053959:Alpha-Amanitin / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000502:physiology*; D006860:Hydrogen Bonding; D007930:Leucine / Q000737:chemistry; D008954:Models, Biological; D008968:Molecular Conformation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012319:RNA Polymerase II / Q000378:metabolism / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014158:Transcription, Genetic
D053959:Alpha-Amanitin; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D007930:Leucine