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References listed in PDB file
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Key reference
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Title
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Sequential structures provide insights into the fidelity of RNA replication.
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Authors
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C.Ferrer-Orta,
A.Arias,
R.Pérez-Luque,
C.Escarmís,
E.Domingo,
N.Verdaguer.
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Ref.
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Proc Natl Acad Sci U S A, 2007,
104,
9463-9468.
[DOI no: ]
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PubMed id
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Abstract
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RNA virus replication is an error-prone event caused by the low fidelity of
viral RNA-dependent RNA polymerases. Replication fidelity can be decreased
further by the use of mutagenic ribonucleoside analogs to a point where viral
genetic information can no longer be maintained. For foot-and-mouth disease
virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be
mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we
report the x-ray structure of four elongation complexes of foot-and-mouth
disease virus polymerase 3D obtained in presence of natural substrates, ATP and
UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine
triphosphate with different RNAs as template-primer molecules. The ability of
these complexes to synthesize RNA in crystals allowed us to capture different
successive replication events and to define the critical amino acids involved in
(i) the recognition and positioning of the incoming nucleotide or analog; (ii)
the positioning of the acceptor base of the template strand; and (iii) the
positioning of the 3'-OH group of the primer nucleotide during RNA replication.
The structures identify key interactions involved in viral RNA replication and
provide insights into the molecular basis of the low fidelity of viral RNA
polymerases.
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Figure 1.
Fig. 1. Structure of FMDV 3D catalytic complexes. Molecular
surface of the polymerase (gray) is shown, with the position of
the rNTP substrates and the trajectory of RNA template–primer
and duplex product in two different complexes: the
3D·GCAUGGGCCC·ATP/UTP (A) and the
3D·GCAUGGGCCC-RTP (B). The N-terminal residues (residues
34–48) and residues at the top of the NTP tunnel (163–180)
of 3D are omitted to show the substrate cavities. RNA molecules
are shown in yellow (template strands) and green (primer
strands). (A) The UTP substrate is shown in cyan. (B) The
position of the antiviral mutagen RTP is shown in orange. Metal
ions are shown as red spheres.
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Figure 2.
Fig. 2. Conserved interactions between the FMDV 3D and the
different RNA template–primers. The polymerase regions
involved in contacts with the RNA molecule are explicitly
labeled. The template and primer strands of the RNA molecule are
shown in yellow and green, respectively; atoms are displayed in
atom-type code, and hydrogen bonds are dashed lines in black.
The template strand contacts mainly with residues in the fingers
subdomain (blue). The 5' overhang region of the template binds
the template channel, where the different residues of the
N-terminal region and the loop  4–  3 of the
polymerase drive the ssRNA to the active-site cavity (Lower
Left). The template strand of the dsRNA product contacts
different residues of helix 7 and the loop 9– 11 in
its exit through the central cavity of the enzyme (Upper Left).
The primer strand interacts with motifs C and E of the palm
subdomain, shown in magenta (Upper Right) and with helix 14 of
the thumb, shown in red (Lower Right).
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Secondary reference #1
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Title
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The structure of a protein primer-Polymerase complex in the initiation of genome replication.
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Authors
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C.Ferrer-Orta,
A.Arias,
R.Agudo,
R.Pérez-Luque,
C.Escarmís,
E.Domingo,
N.Verdaguer.
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Ref.
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EMBO J, 2006,
25,
880-888.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1 Structure of the primer protein VPg in a complex with
3D. (A) Stereo view of a sigma A weighted |F[o]|-|F[c]| electron
density map at 2.9 Å resolution and contoured at 3.0  around
the VPg-UMP molecule (The VPg-UMP and ions were omitted from the
phasing model). The 15 amino acids of VPg, the UMP covalently
linked to the protein and the metal ions are placed inside the
density in ball and stick representation colored in atom type
code. Names for all residues are explicitly labeled in one
letter code. (B) Details of the interactions seen in the active
site of the 3D polymerase during the uridylylation reaction. The
residues Pro2, Tyr3 and Ala4 of VPg are shown in sticks in red
and the UMP, covalently linked to the hydroxyl group of Tyr3, in
light green. The divalent cations Mn2+ and Mg2+ are shown as
magenta and orange spheres, respectively, and the anomalous
difference Fourier map is shown as a chicken wire in blue. The
3D amino acids involved in direct hydrogen bonds with ions and
the uridylylated tyrosine are shown in ball and sticks in atom
type code, and the hydrogen bonds appear as dashed lines. All
residues are explicitly labeled. The predicted position of the
oligo(A) template strand (dark green) was determined using the
3D-RNA template-primer complex (PDB entry 1WNE) as a guide.
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Figure 3.
Figure 3 VPg-3D polymerase interactions. (A) Structure of the
VPg primer protein (red) with the contacting residues of the 3D
polymerase shown in different colors. Four different regions of
the polymerase molecule contact VPg residues E166, I167, R168,
K172 and R179, belonging to motif F of fingers (orange),
together with residues T407, A410 and I411 of the thumb domain
(light blue), interact with the N-terminal moiety of VPg,
stabilizing the conformation of Y3 in the active site cavity. In
addition, residues E166, I167 of motif F (orange), K387 and R388
of motif E (dark blue) and T407, A410 and I411 of helix  13
(light blue) interact with the central part of the VPg protein.
Finally, the 3D residues G216, C217 and P219, located at the
beginning of helix 8
(light blue) in the fingers domain, together with the side chain
of Y336 within the C motif (yellow) of the palm domain,
establish hydrophobic contacts with R11 at the exit of the
polymerase cavity. (B) Structure of the uridylylated VPg protein
(shown in red and the linked UMP in green) with the contacting
residues of the 3D polymerase shown in blue. In addition to the
interactions described in (A), amino acids D245 of motif A
(pink) and D338 of motif C (yellow) are placed in the correct
orientation for the catalysis of the phosphodiester linkage in
the active site of the 3D protein.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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Secondary reference #2
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Title
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A comparison of viral RNA-Dependent RNA polymerases.
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Authors
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C.Ferrer-Orta,
A.Arias,
C.Escarmís,
N.Verdaguer.
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Ref.
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Curr Opin Struct Biol, 2006,
16,
27-34.
[DOI no: ]
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PubMed id
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Secondary reference #3
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Title
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Structure of foot-And-Mouth disease virus RNA-Dependent RNA polymerase and its complex with a template-Primer RNA.
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Authors
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C.Ferrer-Orta,
A.Arias,
R.Perez-Luque,
C.Escarmís,
E.Domingo,
N.Verdaguer.
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Ref.
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J Biol Chem, 2004,
279,
47212-47221.
[DOI no: ]
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PubMed id
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Figure 3.
FIG. 3. Electron density maps around the FMDV 3D active
site. A, stereoview of the final  [A]-weighted 2F[o] -
F[c] Fourier map, contoured at 1.5 , in the isolated 3D
structure with the model placed inside (ball and sticks colored
in atom type code). B, [A]-weighted 2F[o] -
F[c] map, shown at 1.0 , in the FMDV 3D-RNA
complex structure. A portion of the RNA oligonucleotide is shown
in the picture in a stick representation in light green, the
template strand, and dark green, the primer strand. Only two
nucleotides of the template and three of the primer are shown
for clarity. The Mg2+ ion, located close to acidic residues
Asp238, Asp240, and Asp339, is shown as an orange ball. Water
molecules are shown as red balls and labeled as w.
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Figure 6.
FIG. 6. Protein-protein interactions in FMDV 3D polymerase.
A, ribbon representation of the largest interface of
interactions in the P4[1]2[1]2 crystal lattice, also conserved
in P3[2]21 crystals; B, close up of the interacting surfaces.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #4
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Title
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Mutant viral polymerase in the transition of virus to error catastrophe identifies a critical site for RNA binding.
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Authors
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A.Arias,
R.Agudo,
C.Ferrer-Orta,
R.Pérez-Luque,
A.Airaksinen,
E.Brocchi,
E.Domingo,
N.Verdaguer,
C.Escarmís.
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Ref.
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J Mol Biol, 2005,
353,
1021-1032.
[DOI no: ]
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PubMed id
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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