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PDBsum entry 2r92
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Transferase/RNA
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PDB id
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2r92
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Contents |
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1422 a.a.
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1112 a.a.
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267 a.a.
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178 a.a.
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214 a.a.
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88 a.a.
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171 a.a.
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135 a.a.
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116 a.a.
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65 a.a.
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112 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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Molecular basis of RNA-Dependent RNA polymerase ii activity.
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Authors
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E.Lehmann,
F.Brueckner,
P.Cramer.
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Ref.
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Nature, 2007,
450,
445-449.
[DOI no: ]
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PubMed id
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Abstract
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RNA polymerase (Pol) II catalyses DNA-dependent RNA synthesis during gene
transcription. There is, however, evidence that Pol II also possesses
RNA-dependent RNA polymerase (RdRP) activity. Pol II can use a homopolymeric RNA
template, can extend RNA by several nucleotides in the absence of DNA, and has
been implicated in the replication of the RNA genomes of hepatitis delta virus
(HDV) and plant viroids. Here we show the intrinsic RdRP activity of Pol II with
only pure polymerase, an RNA template-product scaffold and nucleoside
triphosphates (NTPs). Crystallography reveals the template-product duplex in the
site occupied by the DNA-RNA hybrid during transcription. RdRP activity resides
at the active site used during transcription, but it is slower and less
processive than DNA-dependent activity. RdRP activity is also obtained with part
of the HDV antigenome. The complex of transcription factor IIS (TFIIS) with Pol
II can cleave one HDV strand, create a reactive stem-loop in the hybrid site,
and extend the new RNA 3' end. Short RNA stem-loops with a 5' extension suffice
for activity, but their growth to a critical length apparently impairs
processivity. The RdRP activity of Pol II provides a missing link in molecular
evolution, because it suggests that Pol II evolved from an ancient replicase
that duplicated RNA genomes.
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Figure 2.
Figure 2: Crystal structure of a Pol II–RdRP complex. a,
Ribbon model of Pol II (grey) with an initial, unbiased
difference Fourier electron-density map (cyan, contoured at 2.2
 ).
The map was calculated from protein model phases. It reveals the
RNA template–product duplex of scaffold RdRP-ss6
(Supplementary Fig. 2) in the active-centre cleft of Pol II. The
bridge helix is in green. The catalytic metal ion A is depicted
as a magenta sphere, and Zn^2+ ions as cyan spheres. The view is
related to that in Fig. 1a by a 90° rotation around a
vertical axis. b, Comparison of the RNA template–product
duplex in the RdRP EC with the DNA–RNA hybrid duplex in the
transcription EC^7. Protein structures were superimposed by
fitting the active-site aspartate loops.
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Figure 4.
Figure 4: Mechanism of HDV replication initiation. a,
HDV-derived terminal stem-loops consisting of 5 or 6 bp enable
templated incorporation of the next nucleotide(s). b, Pure Pol
II–TFIIS complex cleaves the HDV antigenome terminal segment
and elongates the newly formed 3' end on the addition of NTP.
For cleavage, Pol II–scaffold complex (625 nM) was incubated
with TFIIS (1.25  M)
in transcription buffer for 60 min at 28 °C. For elongation
of the cleavage product, the reaction mixture was incubated with
1 mM NTPs at 28 °C for 20 min. For lane 5, the cleavage
reaction was stopped after 60 min ('Stop'). c, Difference
electron density omit map for the 6-bp HDV stem-loop bound to
the hybrid site of Pol II (calculated with protein phases only,
contoured at 3.0 ).
The disordered loop is indicated with a dashed line. The view is
as in Fig. 2a. d, Superposition of the RNA template–product
duplex in the HDV EC and the RdRP EC (Fig. 2) on the DNA–RNA
hybrid duplex in the transcription EC^7. Protein structures were
superimposed by fitting the active-site aspartate loops. e,
Model of initial interaction of the HDV antigenome terminal
segment with the Pol II–TFIIS complex. The stem-loop is placed
in accordance with the crystal structure (c, d) and the
downstream duplex in accordance with the location of the FC* RNA
3' stem. We predict that the HDV bulge passes the bridge helix
and active site, where cleavage occurs.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2007,
450,
445-449)
copyright 2007.
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