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PDBsum entry 2drb
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Transferase/RNA
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PDB id
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2drb
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References listed in PDB file
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Key reference
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Title
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Complete crystallographic analysis of the dynamics of cca sequence addition.
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Authors
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K.Tomita,
R.Ishitani,
S.Fukai,
O.Nureki.
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Ref.
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Nature, 2006,
443,
956-960.
[DOI no: ]
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PubMed id
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Abstract
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CCA-adding polymerase matures the essential 3'-CCA terminus of transfer RNA
without any nucleic-acid template. However, it remains unclear how the correct
nucleotide triphosphate is selected in each reaction step and how the
polymerization is driven by the protein and RNA dynamics. Here we present
complete sequential snapshots of six complex structures of CCA-adding enzyme and
four distinct RNA substrates with and without CTP (cytosine triphosphate) or ATP
(adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to
force the discriminator nucleoside into the active-site pocket, and then tracks
back after incorporation of the first cytosine monophosphate (CMP).
Accommodation of the second CTP clamps the catalytic cleft, inducing a
reorientation of the turn, which flips C74 to allow CMP to be accepted. In
contrast, after the second CMP is added, the polymerase and RNA primer are
locked in the closed state, which directs the subsequent A addition. Between the
CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes
markedly; this is controlled by the global motion of the enzyme and position of
the primer terminus, and is likely to achieve the CTP/ATP discrimination,
depending on the polymerization stage. Throughout the CCA-adding reaction, the
enzyme tail domain firmly anchors the TPsiC-loop of the tRNA, which ensures
accurate polymerization and termination.
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Figure 2.
Figure 2: Expansion and contraction of the primer RNA helix at
the mini-D stage. a, Extended mini-helix structure at the
mini-D stage. The simulated annealed omit maps (contoured at 3.5
 )
for the indicated nucleosides are shown. b, Back-tracked
mini-helix structure at the mini-DC. Other stages have the same
standard helix structure.
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Figure 3.
Figure 3: Active-site structure at each reaction stage. a,
Mini-D stage (the simulated annealed omit maps contoured at 4
for
G1 and A73 are shown). b, Mini-DC stage. c, Mini-DC + CTP stage.
d, Mini-DCC stage. e, Mini-DCC + ATP stage. f, Mini-DCCA stage.
g, Mature tRNA dissociation stage (PDB ID: 1SZ1)^14. The
acceptor stem expansion at the first CCA-adding step can easily
be seen by the fact that Asp 291 recognizes the 2'-OH group of
C72 in the mini-D stage, but A73 in the other stages. The
catalytic triad comprises Glu 59, Asp 61 and Asp 110. The
hydrogen bonds are represented by dotted lines.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2006,
443,
956-960)
copyright 2006.
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