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PDBsum entry 2q66
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Transferase/RNA
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PDB id
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2q66
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References listed in PDB file
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Key reference
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Title
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Mechanism of poly(a) polymerase: structure of the enzyme-Mgatp-Rna ternary complex and kinetic analysis.
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Authors
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P.B.Balbo,
A.Bohm.
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Ref.
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Structure, 2007,
15,
1117-1131.
[DOI no: ]
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PubMed id
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Abstract
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We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in
complex with ATP and a five residue poly(A) by mutation of the catalytically
required aspartic acid 154 to alanine. The enzyme has undergone significant
domain movement and reveals a closed conformation with extensive interactions
between the substrates and all three polymerase domains. Both substrates and 31
buried water molecules are enclosed within a central cavity that is open at both
ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies
of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and
nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The
results support a model in which binding of both poly(A) and the correct
nucleotide, MgATP, induces a conformational change, resulting in formation of a
stable, closed enzyme state. Thermodynamic considerations of the data are
discussed as they pertain to domain closure, substrate specificity, and
catalytic strategies utilized by PAP.
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Figure 2.
Figure 2. Determinants of ATP and RNA Binding
Interacting residues from the N-terminal, middle, and C-terminal
domains are colored yellow, green, and cyan, respectively.
Residues within cloud-shaped bubbles on either side of the bases
indicate hydrophobic/van der Waals interactions. Water molecules
(circles) are color coded based on the degree to which they are
buried within the interior of the protein; those colored dark
blue are completely buried (see Experimental Procedures). Those
colored gray-blue are at the protein surface. Others are colored
intermediate shades of blue depending on how many shells of
water needed to be removed in order for the water atoms to
become exposed. Asp154 and the second Mg^2+ ion are shown in
gray. Eight additional water molecules interact with the
triphosphate moiety of the ATP. These are not shown for clarity,
and because the water structure in this region may be altered
due to the D154A mutation. The base at position −5 interacts
with a neighboring PAP molecule and is not shown.
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Figure 3.
Figure 3. Stereo Views of the Substrate Binding Sites of PAP
Detailed substrate interactions formed in the closed,
ternary complex. ATP (yellow carbons) and the 3′ end (blue
carbons) are shown along with PAP with the surrounding amino
acids (green carbons) and water molecules (red spheres).
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The above figures are
reprinted
from an Open Access publication published by Cell Press:
Structure
(2007,
15,
1117-1131)
copyright 2007.
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