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PDBsum entry 2hzc
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RNA binding protein
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PDB id
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2hzc
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References listed in PDB file
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Key reference
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Title
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Alternative conformations at the rna-Binding surface of the n-Terminal u2af(65) rna recognition motif.
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Authors
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K.R.Thickman,
E.A.Sickmier,
C.L.Kielkopf.
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Ref.
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J Mol Biol, 2007,
366,
703-710.
[DOI no: ]
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PubMed id
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Abstract
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The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF(65))
recognizes the polypyrimidine tract (Py-tract) consensus sequence of the
pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of
eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are
often interrupted with purines, yet U2AF(65) must identify these degenerate
Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a
U2AF(65) variant in complex with poly(U) RNA suggested that rearrangement of
flexible side-chains or bound water molecules may contribute to degenerate
Py-tract recognition by U2AF(65). Here, the X-ray structure of the N-terminal
RRM domain of U2AF(65) (RRM1) is described at 1.47 A resolution in the absence
of RNA. Notably, RNA-binding by U2AF(65) selectively stabilizes pre-existing
alternative conformations of three side-chains located at the RNA interface
(Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the
beta2/beta3 strands undergoes a conformational change to interact with the RNA.
These pre-existing alternative conformations may contribute to the ability of
U2AF(65) to recognize a variety of Py-tract sequences. This rare,
high-resolution view of an important member of the RRM class of RNA-binding
domains highlights the role of alternative side-chain conformations in RNA
recognition.
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Figure 1.
Figure 1. Overall structure of U2AF^65-RRM1. Alternative
conformations are shown as ball-and-stick representations with
the major conformation in yellow and the minor conformation in
red. (a) Ribbon diagram of U2AF^65-RRM1 with α-helices in blue
and β-strands in green. Bound PEG MME550 and zinc ions from the
crystallization solution are shown in magenta. (b) View as in
(a), rotated 180° about the y-axis. (c) Backbone trace
representations of U2AF^65-RRM1 (blue) compared with X-ray
structures of other RRMs determined in the absence of RNA,
including the N-terminal RRM of U1A^24 (purple), N and
C-terminal RRMs of Sex-lethal^16 (red and green, respectively),
and N and C-terminal RRMs of hnRNP A1^17 (yellow and cyan,
respectively). The unique α1/β2 loop and β2/β3 loop of
U2AF^65-RRM1 are labeled. Images were created using
MolScript,^25 Bobscript,^26 and Raster3D.^27
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Figure 2.
Figure 2. Comparison of apo- and RNA-bound U2AF^65-RRM1.
U2AF^65-RRM1 in the presence (green) or in the absence (blue) of
RNA ligand. When alternative conformations are present, major
conformations of the apo-RRM1 side-chains are colored yellow,
minor conformations are colored orange. (a) Superimposed ribbon
diagrams. (b) View of the α1/β2 loop. (c) Comparison of
aromatic residues (Phe197, Phe199, and Tyr152) in the RNP
motifs. The σA-weighted, |F[o]|–|F[c]| omit electron density
for side-chain atoms was calculated using Shelxpro^28 and is
shown at the 5σ contour level. (d) View of Arg150. (e) View of
Lys225 and Arg227.
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The above figures are
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
366,
703-710)
copyright 2007.
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Secondary reference #1
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Title
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Structural basis for polypyrimidine tract recognition by the essential pre-Mrna splicing factor u2af65.
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Authors
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E.A.Sickmier,
K.E.Frato,
H.Shen,
S.R.Paranawithana,
M.R.Green,
C.L.Kielkopf.
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Ref.
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Mol Cell, 2006,
23,
49-59.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. Overall Structure of the Human dU2AF^651,2/rU[7]
Complex
(A) View of the crystal packing interactions of
the dU2AF^651,2/rU[7] complex. The symmetry-related protein
molecules are shown as green or blue backbone worms, and the
bound nucleic acids are shown as purple or yellow ball-and-stick
models. One asymmetric unit contains one protein molecule and
one rU[7] oligonucleotide, so that each rU[7] oligonucleotide is
bound by RRM1 and RRM2 donated by distinct, symmetry-related
protein molecules.
(B) View of the integrated structural
unit of RRM1 and RRM2 interacting with the rU[7] strand, into
the RNA binding surface (top panel) or rotated 180° about
the vertical axis (lower panel). Secondary structure elements of
the dU2AF^651,2 protein are labeled (primed italics
distinguish RRM2 secondary structures) on a ribbon model that is
colored by domain as follows: RRM1 (residues 148–237), green;
and RRM2 (residues 258–336), blue. A composite omit electron
density map at 1σ contour level surrounds a stick
representation of the oligonucleotides (purple).
(C)
Electrostatic surface representation of dU2AF^651,2, shown bound
to a stick representation of the RNA in a similar orientation as
the lower panel of (B) was generated using the program Pymol
(http://www.pymol.org). Red areas, highly negatively charged
residues; blue areas, highly positively charged residues.
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Figure 5.
Figure 5. Uridine Recognition in the dU2AF^651,2/rU[7]
Complex
(A) Schematic diagram of dU2AF^651,2/rU[7]
interactions. Residue names are boxed and colored as in
(B)–(H). Filled boxes indicate main chain contacts, and open
boxes indicate side chain interactions. Dashed lines represent
hydrogen bonds, dashed lines ending with a filled circle
represent stacking interactions, and solid lines represent ionic
interactions.
(B–H) Ball-and-stick representations of the
interactions between dU2AF^651,2 and each bound uridine are
shown. Residues belonging to RRM1 are colored green, residues
from RRM2 are colored blue, and residues altered by
site-directed mutagenesis are colored yellow. RNA bonds are
colored purple, with a lighter shade distinguishing the C5-C6
bond of the base edges. The oxygen atoms of the stick
representations are colored red, and nitrogen atoms are blue.
Dashed lines indicate hydrogen bonds. Recognition of (B) Uri1,
(C) Uri2, (D) Uri3, (E) Uri4, (F) Uri5, (G) Uri6, and (H) Uri7.
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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