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References listed in PDB file
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Key reference
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Title
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Unravelling the dynamics of RNA degradation by ribonuclease ii and its RNA-Bound complex.
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Authors
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C.Frazão,
C.E.Mcvey,
M.Amblar,
A.Barbas,
C.Vonrhein,
C.M.Arraiano,
M.A.Carrondo.
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Ref.
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Nature, 2006,
443,
110-114.
[DOI no: ]
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PubMed id
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Abstract
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RNA degradation is a determining factor in the control of gene expression. The
maturation, turnover and quality control of RNA is performed by many different
classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease
that intervenes in all of these fundamental processes; it can act independently
or as a component of the exosome, an essential RNA-degrading multiprotein
complex. RNase II-like enzymes are found in all three kingdoms of life, but
there are no structural data for any of the proteins of this family. Here we
report the X-ray crystallographic structures of both the ligand-free (at 2.44 A
resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase
II. In contrast to sequence predictions, the structures show that RNase II is
organized into four domains: two cold-shock domains, one RNB catalytic domain,
which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme
establishes contacts with RNA in two distinct regions, the 'anchor' and the
'catalytic' regions, which act synergistically to provide catalysis. The active
site is buried within the RNB catalytic domain, in a pocket formed by four
conserved sequence motifs. The structure shows that the catalytic pocket is only
accessible to single-stranded RNA, and explains the specificity for RNA versus
DNA cleavage. It also explains the dynamic mechanism of RNA degradation by
providing the structural basis for RNA translocation and enzyme processivity. We
propose a reaction mechanism for exonucleolytic RNA degradation involving key
conserved residues. Our three-dimensional model corroborates all existing
biochemical data for RNase II, and elucidates the general basis for RNA
degradation. Moreover, it reveals important structural features that can be
extrapolated to other members of this family.
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Figure 2.
Figure 2: RNase II–ssRNA interactions.
Figure 2 :
RNase II|[ndash]|ssRNA interactions. Unfortunately we are
unable to provide accessible alternative text for this.
If you require assistance to access this image, or to
obtain a text description, please contact npg@nature.com-
a, Atomic interactions scheme between RNA (enclosed atoms in
black or in red for O2'-protein interactions) and protein
residues (coloured by domains as in Fig. 1a). Hb indicates
hydrogen bonds up to 3.2 Å, and vdW indicates van der
Waals interactions up to 3.6 Å. b, c, RNA docking
catalytic region (b) with residues 364–381 deleted for
clarity, and anchor region (c) involving canonical
oligonucleotide-binding motifs^16 L[23] of CSD2, and chain  3
and loop L[45] of S1. RNA (coloured as in Fig. 1) and protein
hydrogen-bonding side-chain residues shown in sticks (carbon in
yellow, nitrogen in cyan, oxygen in red), enzyme domains as
cartoons (coloured as in Fig. 1). Hydrogen bonds shown as dashed
green lines, Mg shown as green sphere with coordination in
dashed yellow lines. Nucleotides (Nt) 9–13 are clamped between
the aromatic side-chains of Tyr 253 and Phe 358 (with labels
highlighted in green), nucleotides 3 and 4 are between Phe 588
and Pro 104, and nucleotide 5 is nestled between His 103, Pro
104, Asp 102 and Arg 167.
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Figure 3.
Figure 3: RNA degradation by RNase II.
Figure 3 : RNA
degradation by RNase II. Unfortunately we are unable to
provide accessible alternative text for this. If you
require assistance to access this image, or to obtain a
text description, please contact npg@nature.com-
a, Stereo view of RNase II D209N mutant active site; bonds
are shown as sticks (oxygen in red, nitrogen in blue, phosphorus
in orange), waters as red spheres, Mg as a green sphere and its
coordination as dashed orange lines, and hydrogen bonds as
dashed green lines, superposed with sigma-A corrected Fourier
synthesis electron density map (brown mesh) contoured at 1  .
Additionally, N1 and N6 of nucleotide 13 are in the vicinity of
the carboxylate oxygens of Glu 542, at 3.2 Å (hydrogen
bond as dashed green lines) and at 4.3 Å (distance as
dotted orange line), respectively. b, Magnified view of the Mg
ion and its coordinating environment (distances in Å)
superposed with the positive 3 sigma-A
corrected Fourier difference map (green mesh) calculated with
the Mg and coordinating waters omitted from the model. c, Stereo
view of the superposition of the RNase II D209N mutant (yellow)
and RNase H (grey) with magnesium coordinating spheres (opposite
view to Fig. 3a). RNase H displays two magnesium ions ligated by
Glu 109, D132N, Glu 188 and Asp 192 that correspond in RNase II
to Asp 201, Asp 210, D209N and Asp 207, respectively. d,
Proposed catalytic mechanism for RNase II, showing the
postulated second Mg (Mg II) and the attacking hydroxyl group
(grey). e, Model for RNA degradation by RNase II. ssRNA (red) is
threaded into the catalytic cavity and clamped between Tyr 253
and Phe 358. The additional stabilization of RNA inside the
cavity drives the RNA translocation after each cleavage, up to a
final four-nucleotide fragment.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2006,
443,
110-114)
copyright 2006.
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Secondary reference #1
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Title
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Expression, Purification, Crystallization and preliminary diffraction data characterization of escherichia coli ribonuclease ii (rnase ii).
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Authors
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C.E.Mcvey,
M.Amblar,
A.Barbas,
F.Cairrão,
R.Coelho,
C.Romão,
C.M.Arraiano,
M.A.Carrondo,
C.Frazão.
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Ref.
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Acta Crystallograph Sect F Struct Biol Cryst Commun, 2006,
62,
684-687.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3 RNase II D209N SIRAS statistics. (a) As a zero signal,
is about 0.8 (SHELXC output documentation), the SeMet
diffraction data (blue) is shown to contain anomalous signal
until its resolution cutoff, in contrast to the mutant native
data (red). (b) The Se site-occupancy profile indicates that the
asymmetric unit contains only one RNase II molecule, as RNase II
contains 16 Met residues in its sequence and residual
occupancies of 0.2 indicate noise peaks (Schneider & Sheldrick,
2002[Schneider, T. R. & Sheldrick, G. M. (2002). Acta Cryst.
D58, 1772-1779.]). (c) The space-group ambiguity was solved by
SHELXE as the density-modification `map contrast' statistics
(Sheldrick, 2002[Sheldrick, G. M. (2002). Z. Kristallogr. 217,
644-650.]) assuming space group P6[5] (red) clearly converged to
a higher value when compared with that of its hand-inverted
counterpart P6[1] (blue).
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The above figure is
reproduced from the cited reference
which is an Open Access publication published by the IUCr
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Secondary reference #2
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Title
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Characterization of the functional domains of escherichia coli rnase ii.
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Authors
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M.Amblar,
A.Barbas,
A.M.Fialho,
C.M.Arraiano.
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Ref.
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J Mol Biol, 2006,
360,
921-933.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Schematic representation of RNase II and the deletion
derivatives constructed. The three putative domains predicted
for E. coli RNase II are represented as rectangles: CSD (gray),
RNB domain (white) and S1 domain (diagonal stripes), and a
black box at the N terminus of each enzyme represents the
His-tag fused. The amino acids at the limits of each domain are
indicated. For amino acid numbering, the sequence used as
template was the untagged wild-type RNase II. The four highly
conserved sequence motifs of the RNB domain (motif I to IV) are
also depicted. Restriction sites used for the mutant
construction are indicated, and those introduced by mutagenesis
are labeled with an asterisk (*). The region removed on each
deletion mutant is represented as a broken line and the
numbering corresponds to the residues limiting the deleted
region. On the right, the plasmid encoding each deletion mutant
is indicated, as well as the molecular mass of the mutant
protein and their solubility after IPTG induction: more than 50%
soluble (S), less than 50% soluble (I). On the left is shown
the name used for each deletion protein. Figure 1. Schematic
representation of RNase II and the deletion derivatives
constructed. The three putative domains predicted for E. coli
RNase II are represented as rectangles: CSD (gray), RNB domain
(white) and S1 domain (diagonal stripes), and a black box at the
N terminus of each enzyme represents the His-tag fused. The
amino acids at the limits of each domain are indicated. For
amino acid numbering, the sequence used as template was the
untagged wild-type RNase II. The four highly conserved sequence
motifs of the RNB domain (motif I to IV) are also depicted.
Restriction sites used for the mutant construction are
indicated, and those introduced by mutagenesis are labeled with
an asterisk (*). The region removed on each deletion mutant is
represented as a broken line and the numbering corresponds to
the residues limiting the deleted region. On the right, the
plasmid encoding each deletion mutant is indicated, as well as
the molecular mass of the mutant protein and their solubility
after IPTG induction: more than 50% soluble (S), less than 50%
soluble (I). On the left is shown the name used for each
deletion protein.
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Figure 6.
Figure 6. Analysis of RNB amino acid sequence. (a) Schematic
representation of the domains found in prokaryotic
exoribonuclease II: CSD, cold shock domain; RNB, conserved
central region of about 400 amino acid residues that contains
four sequence motifs (I–IV); S1, RNA-binding domain. (b)
Multiple amino acid sequence alignment (CLUSTAL X^43) of the
residues comprising the motifs I to IV of the RNB domain (upper
part) and the N-terminal part of the RNB domain (*) containing a
new conserved region (lower part). The consensus sequence is
represented bellow the alignment. Bacterial genus and species
are abbreviated as follows: Esco, Escherichia coli K12 (acession
number: NP_415802.1); Vich, Vibrio cholerae O395
(ZP_00754758.1); Vivu, Vibrio vulnificus CMCP6 (NP_762129.1);
Shfl, Shigella flexneri 2a str. 2457T (NP_836978.1); Saty,
Salmonella typhimurium LT2 (AAL20620.1); Yepe, Yersinia pestis
biovar Medievalis str. 91001 (AAS62250.1); Erca, Erwinia
carotovora subsp. atroseptica SCRI1043 (CAG74865.1); Phlu,
Photorhabdus luminescens subsp. laumondii TTO1 (NP_929629.1);
Hain, Haemophilus influenzae Rd KW20 (NP_439875.1); Haso,
Haemophilus somnus 2336 (ZP_00133292.2); Pamu, Pasteurella
multocida subsp. multocida str. Pm70 (AAK02265.1); Acpl,
Actinobacillus pleuropneumoniae serovar 1 str. 4074
(ZP_00134345.1). Numbers of intervening amino acids are given in
brackets. (c) Secondary structure prediction of the N-terminal
part (residues from 85 to 156) of the RNB domain of E.coli RNase
II. β-Sheet elements are illustrated as arrows. The secondary
structure prediction was made using 3D-PSSM program, Folding
Recognition Server. Figure 6. Analysis of RNB amino acid
sequence. (a) Schematic representation of the domains found in
prokaryotic exoribonuclease II: CSD, cold shock domain; RNB,
conserved central region of about 400 amino acid residues that
contains four sequence motifs (I–IV); S1, RNA-binding domain.
(b) Multiple amino acid sequence alignment (CLUSTAL X[3]^43) of
the residues comprising the motifs I to IV of the RNB domain
(upper part) and the N-terminal part of the RNB domain (*)
containing a new conserved region (lower part). The consensus
sequence is represented bellow the alignment. Bacterial genus
and species are abbreviated as follows: Esco, Escherichia coli
K12 (acession number: NP_415802.1); Vich, Vibrio cholerae O395
(ZP_00754758.1); Vivu, Vibrio vulnificus CMCP6 (NP_762129.1);
Shfl, Shigella flexneri 2a str. 2457T (NP_836978.1); Saty,
Salmonella typhimurium LT2 (AAL20620.1); Yepe, Yersinia pestis
biovar Medievalis str. 91001 (AAS62250.1); Erca, Erwinia
carotovora subsp. atroseptica SCRI1043 (CAG74865.1); Phlu,
Photorhabdus luminescens subsp. laumondii TTO1 (NP_929629.1);
Hain, Haemophilus influenzae Rd KW20 (NP_439875.1); Haso,
Haemophilus somnus 2336 (ZP_00133292.2); Pamu, Pasteurella
multocida subsp. multocida str. Pm70 (AAK02265.1); Acpl,
Actinobacillus pleuropneumoniae serovar 1 str. 4074
(ZP_00134345.1). Numbers of intervening amino acids are given in
brackets. (c) Secondary structure prediction of the N-terminal
part (residues from 85 to 156) of the RNB domain of E.coli RNase
II. β-Sheet elements are illustrated as arrows. The secondary
structure prediction was made using 3D-PSSM program, Folding
Recognition Server. [4]^44
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #3
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Title
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Dna sequencing and expression of the gene rnb encoding escherichia coli ribonuclease ii.
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Authors
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R.Zilhão,
L.Camelo,
C.M.Arraiano.
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Ref.
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Mol Microbiol, 1993,
8,
43-51.
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PubMed id
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