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PDBsum entry 2d0c
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References listed in PDB file
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Key reference
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Title
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Crystal structure and structure-Based mutational analyses of rnase hiii from bacillus stearothermophilus: a new type 2 rnase h with tbp-Like substrate-Binding domain at the n terminus.
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Authors
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H.Chon,
H.Matsumura,
Y.Koga,
K.Takano,
S.Kanaya.
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Ref.
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J Mol Biol, 2006,
356,
165-178.
[DOI no: ]
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PubMed id
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Abstract
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Ribonuclease HIII (Bst-RNase HIII) from the moderate thermophile Bacillus
stearothermophilus is a type 2 RNase H but shows poor amino acid sequence
identity with another type 2 RNase H, RNase HII. It is composed of 310 amino
acid residues and acts as a monomer. Bst-RNase HIII has a large N-terminal
extension with unknown function and a unique active-site motif (DEDE), both of
which are characteristics common to RNases HIII. To understand the role of these
N-terminal extension and active-site residues, the crystal structure of
Bst-RNase HIII was determined in both metal-free and metal-bound forms at
2.1-2.6 angstroms resolutions. According to these structures, Bst-RNase HIII
consists of the N-terminal domain and C-terminal RNase H domain. The structures
of the N and C-terminal domains were similar to those of TATA-box binding
proteins and archaeal RNases HII, respectively. The steric configurations of the
four conserved active-site residues were very similar to those of other type 1
and type 2 RNases H. Single Mn and Mg ions were coordinated with Asp97, Glu98,
and Asp202, which correspond to Asp10, Glu48, and Asp70 of Escherichia coli
RNase HI, respectively. The mutational studies indicated that the replacement of
either one of these residues with Ala resulted in a great reduction of the
enzymatic activity. Overproduction, purification, and characterization of the
Bst-RNase HIII derivatives with N and/or C-terminal truncations indicated that
the N-terminal domain and C-terminal helix are involved in substrate binding,
but the former contributes to substrate binding more greatly than the latter.
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Figure 4.
Figure 4. Stereo view of electron density around the active
site of Bst-RNase HIII. The structures of (a) metal-free, ((b)
and (d)) Mn2+-bound, and (c) Mg2+-bound proteins are shown. In
(a), (b), and (c), the 2F[o] -F[c] map contoured at the 1.0s
level is shown. In (d), the 2F[o] -F[c] map and the anomalous
difference map contoured at the 4.5s and 3.5s levels are shown
in blue and magenta, respectively. The red and yellow crosses
indicate the water molecule and metal ion, respectively.
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Figure 5.
Figure 5. The structures of the active sites of various
RNases H. The side-chains of the active-site residues in the
crystal structures of metal-free (green), Mn2+-bound (blue), and
Mg2+-bound (magenta) Bst-RNases HIII, Tk-RNase HII (red), and E.
coli RNase HI (cyan) are shown. The view direction is the same
as in 1 and 3.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
356,
165-178)
copyright 2006.
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