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PDBsum entry 1wrn
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RNA binding protein
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PDB id
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1wrn
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References listed in PDB file
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Key reference
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Title
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Characterization of the metal ion binding site in the anti-Terminator protein, Hutp, Of bacillus subtilis.
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Authors
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T.Kumarevel,
H.Mizuno,
P.K.Kumar.
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Ref.
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Nucleic Acids Res, 2005,
33,
5494-5502.
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PubMed id
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Abstract
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HutP is an RNA-binding protein that regulates the expression of the histidine
utilization (hut) operon in Bacillus subtilis, by binding to cis-acting
regulatory sequences on hut mRNA. It requires L-histidine and an Mg2+ ion for
binding to the specific sequence within the hut mRNA. In the present study, we
show that several divalent cations can mediate the HutP-RNA interactions. The
best divalent cations were Mn2+, Zn2+ and Cd2+, followed by Mg2+, Co2+ and Ni2+,
while Cu2+, Yb2+ and Hg2+ were ineffective. In the HutP-RNA interactions,
divalent cations cannot be replaced by monovalent cations, suggesting that a
divalent metal ion is required for mediating the protein-RNA interactions. To
clarify their importance, we have crystallized HutP in the presence of three
different metal ions (Mg2+, Mn2+ and Ba2+), which revealed the importance of the
metal ion binding site. Furthermore, these analyses clearly demonstrated how the
metal ions cause the structural rearrangements that are required for the hut
mRNA recognition.
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Figure 5.
Divalent metal ion coordinations in the complex structures. (A) A close up
stereo view of the Ba^2+ ion binding site in the HutP-L-histidine-Ba^2+ complex. Hydrogen
bonds are indicated by broken lines. The L-histidine ligand and the protein residues are
represented by ball-and-stick models colored by atom type, as shown in Figure 4c. The
Ba^2+ and water molecules are represented by cpk models in magenta and red, respectively.
The electron density around the metal ion was contoured at 3 {sigma} level. (B) A
close-up stereo view of the non-specific Ba^2+ ion binding site and its interactions. The
electron density around the metal ions was contoured at 3 {sigma} level. Hydrogen
bonds and the color scheme are described in Figure 5a.
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Figure 6.
Divalent metal ion coordination distance comparison for different metal ions
observed in the complex structures. A schematic hexa-coordination of the metal ions, drawn
and numbered as in Figure 5a. The metal ion binding sites observed in the asymmetric
unit were averaged individually and are depicted in the figures.
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The above figures are
reprinted
from an Open Access publication published by Oxford University Press:
Nucleic Acids Res
(2005,
33,
5494-5502)
copyright 2005.
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Secondary reference #1
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Title
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Structural basis of hutp-Mediated anti-Termination and roles of the mg2+ ion and l-Histidine ligand.
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Authors
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T.Kumarevel,
H.Mizuno,
P.K.Kumar.
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Ref.
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Nature, 2005,
434,
183-191.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4: Stereo views of conformational changes observed in the
quaternary complex. a, A comparison between the quaternary
complex (blue) and the HutP -HBN complex (red) showing the
structural differences around the l-histidine and Mg2+ binding
site. Symm. related mol., neighbouring dimer within the hexamer.
b, Conformational changes observed in the loop L3 region,
coloured as in a. c, Conformational changes observed in loop L5,
coloured as in a.
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Figure 5.
Figure 5: Electrostatic surface potential models of HutP and the
proposed mechanism for the anti-terminator complex formation.
a -d, Molecular surfaces of the HutP dimer of uncomplexed HutP
(a), HutP -HBN (b), the HutP -l-histidine -Mg2+ complex (c) and
the quaternary HutP complex (d), coloured in accordance with the
electrostatic potential. HBN, l-histidine and RNA are
represented by ball-and-stick models. Mg2+ ions are represented
by a cpk model. e, A schematic model proposed for HutP
anti-terminator complex formation. f, A proposed model for the
existence of two potential binding sites (highlighted in blue
boxes) within the terminator region. The GC-rich region is
highlighted in the red box. The RNA-binding residues are
indicated by magenta and green.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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Secondary reference #2
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Title
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Crystal structure of activated hutp; an RNA binding protein that regulates transcription of the hut operon in bacillus subtilis.
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Authors
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T.Kumarevel,
Z.Fujimoto,
P.Karthe,
M.Oda,
H.Mizuno,
P.K.Kumar.
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Ref.
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Structure, 2004,
12,
1269-1280.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4. Details of the Interactions of HutP-HBN
Complex(A) Molecular surface representation of HutP interacting
with HBN. The surfaces are colored according to the
electrostatic potential (blue, positive; red, negative). The
imidazole ring of HBN is buried within the hydrophobic pocket of
the HutP.(B) A close stereoview of the HBN binding site in HutP.
Hydrogen bonds are indicated by broken lines. HBN is represented
by a ball-and-stick model.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #3
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Title
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Identification of important chemical groups of the hut mRNA for hutp interactions that regulate the hut operon in bacillus subtilis.
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Authors
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T.S.Kumarevel,
S.C.Gopinath,
S.Nishikawa,
H.Mizuno,
P.K.Kumar.
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Ref.
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Nucleic Acids Res, 2004,
32,
3904-3912.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4. Effects of base substitutions and deoxy ribose
substitutions at the UAG motif on HutP binding. (a) Effects of
base substitutions at the UAG motif on HutP binding. RNAs
containing substitutions (A or G or C) were prepared chemically,
labeled at the 5' end, and then used in the filter binding assay
described in Figure 2. (b) Effects of base substitutions and the
base analog at the second base of the UAG motif on HutP binding.
RNAs containing substitutions (U or G or C), in addition to RNA
containing the 2-amino purine analog, were prepared chemically,
labeled at the 5' end, and then used in the filter binding assay
described in Figure 2. (c) Effects of base substitutions and the
base analog at the third base of the UAG motif on HutP binding.
RNAs containing substitutions (A or C or U), in addition to RNA
containing ribo-inosine, were prepared chemically, labeled at
the 5' end, and then used in the filter binding assay described
in Figure 2. To evaluate the positional importance for the UAG
motifs' recognition, we prepared three RNAs containing inosine
substitutions at the first, second and third UAG motif in the
21mer RAT. These RNAs were tested for HutP binding. (d) Effects
of deoxy base substitutions at the UAG motif on HutP binding. To
evaluate the 2'-OH group importance for the UAG motifs'
recognition, three RNAs containing substitutions (dU or dA or
dG), were prepared chemically, labeled at the 5' end, and then
used in the filter binding assay described in Figure 2.
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Figure 6.
Figure 6. Potential RNA binding sites of HutP.
Electrostatic potential of the molecular surface of hexameric
HutP, based on the crystal structure of HutP. The electrostatic
potential was calculated and visualized using GRASP (18). Basic
regions are shown in blue and acidic regions are red. The
electronegative potentials for the Glu55 residues are indicated
by arrows and arrowheads, respectively. The important A and G
bases for the HutP recognition are highlighted with bigger sized
letters.
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by Oxford University Press
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