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PDBsum entry 1vfg
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Transferase/RNA
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PDB id
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1vfg
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Contents |
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* Residue conservation analysis
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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 for template-Independent RNA polymerization.
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Authors
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K.Tomita,
S.Fukai,
R.Ishitani,
T.Ueda,
N.Takeuchi,
D.G.Vassylyev,
O.Nureki.
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Ref.
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Nature, 2004,
430,
700-704.
[DOI no: ]
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PubMed id
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Abstract
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The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is
essential for amino acid attachment and interaction with the ribosome during
protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a
template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as
substrates. Despite structural and biochemical studies, the mechanism by which
the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid
template remains elusive. Here we present the crystal structure of Aquifex
aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal
adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds
the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is
adjacent to ATP, and their base moieties are stacked. The complementary pocket
for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate
two nucleotides, mimicking the nucleotide template used by template-dependent
polymerases. These results are supported by systematic analyses of mutants. Our
structure represents the 'pre-insertion' stage of selecting the incoming
nucleotide and provides the structural basis for the mechanism underlying
template-independent RNA polymerization.
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Figure 2.
Figure 2: Stereoview of the primer C74-C75 and the incoming ATP.
a,  [A]-Weighted
simulated-annealing F[o] - F[c] omit maps contoured at 3.5 around
C74-C75 and AMPcPP. The carbon atoms of Aa.LC, tRNA and AMPcPP
are coloured white, pink and blue, respectively. b, Recognition
of the incoming ATP. Ball-and-stick representations of tRNA C75,
AMPcPP and the ATP-interacting residues are shown on the Aa.LC
head and neck domains. The colouring scheme is the same as in
Fig. 1. c, Recognition of the C74-C75 terminus. Ball-and-stick
representations of tRNA A73-C74-C75, AMPcPP and the
tRNA-interacting residues are shown. Phe 106 and the Asp105
-Arg155 pair, which are part of the 'stacking arc', are also
shown in ball-and-stick representation. In b and c, hydrogen
bonds are shown as dotted lines.
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Figure 4.
Figure 4: Comparison of template-independent and
template-dependent RNA polymerases. a, Ball-and-stick
representations of tRNA, AMPcPP, the catalytic carboxylates and
the ATP-interacting residues are shown on the Aa.LC head and
neck domains. The colouring scheme is the same as in Fig. 2,
except that the carbon atoms of Aa.LC are coloured orange. b,
Ball-and-stick representations of the primer RNA, the template
DNA, AMPcPP, the catalytic carboxylates and the ATP-interacting
residues are shown on the O helix in the T7 RNA polymerase
structure. In a and b, hydrogen bonds are shown as dotted lines.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2004,
430,
700-704)
copyright 2004.
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