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PDBsum entry 2vum
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Contents |
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1418 a.a.
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1106 a.a.
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266 a.a.
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177 a.a.
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214 a.a.
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84 a.a.
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171 a.a.
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133 a.a.
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119 a.a.
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65 a.a.
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114 a.a.
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46 a.a.
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References listed in PDB file
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Key reference
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Title
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Structural basis of transcription inhibition by alpha-Amanitin and implications for RNA polymerase ii translocation.
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Authors
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F.Brueckner,
P.Cramer.
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Ref.
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Nat Struct Biol, 2008,
15,
811-818.
[DOI no: ]
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PubMed id
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Abstract
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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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Figure 4.
(a,b) Superposition of the trigger loops and bridge helices
in the  -amanitin
inhibited Pol II EC and the free T. thermophilus (Tth) RNA
polymerase^6. The trigger loop residue Leu1081 (S. cerevisiae
(Sc) Pol II) or its homologous residue Met1238 (Tth) forms a
wedge between the bridge helix and helix 37
in Pol II or G' in Tth. The views are from the top (a) or the
side (b), as in Figure 3b or 1e, respectively. In the -amanitin–inhibited
Pol II EC, the central bridge helix is shifted, whereas in the
bacterial holoenzyme it adopts a flipped-out conformation. (c,d)
Four possible states of the EC. Above to below, the
pretranslocation state (PDB 1I6H)^1, a potential transition
state with a modeled flipped-out bridge helix (PDB 1IW7)^17, the
-amanitin–inhibited
EC (the apparent translocation intermediate with the shifted
bridge helix, this study), and the post-translocation state (PDB
1Y1W)^2 are shown with space-filling models (c) or ribbon
diagrams (d). The bridge helix residues Ala832/Ala1089 (Pol
II/Tth) and Thr831/Thr1088 (Pol II/Tth) are highlighted in
yellow and brown, respectively. (e,f) Comparison of trigger loop
conformations. Pol II EC structures in the post-translocation
state (PDB 1Y1W)^2, with bound NTP substrate (PDB 2E2H)^4, and
in the intermediary state are superimposed. Nucleic acids and
metal A are from the translocation intermediate. The trigger
loops of the three structures are depicted in dark red (wedged,
translocation intermediate), light blue (open, 1Y1W) and yellow
(closed, 2E2H, labels in black). (f) Also depicted are the
bridge helix (green, apparent translocation intermediate) and
the NTP in the insertion site (orange, 2E2H). (g) Comparison of
bridge helix conformations in the -amanitin–inhibited
EC (green, with residues Ala832 and Thr831 highlighted in yellow
and brown, respectively), the post-translocation EC^2 (light
green) and the core Pol II EC with bound NTP^4 (beige).
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Figure 5.
Schematic representation of the extended model for the NAC.
The vertical dashed line indicates register +1. For details,
refer to text.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Biol
(2008,
15,
811-818)
copyright 2008.
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