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PDBsum entry 2ghp
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RNA binding protein
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PDB id
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2ghp
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References listed in PDB file
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Key reference
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Title
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Structure and interactions of the first three RNA recognition motifs of splicing factor prp24.
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Authors
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E.Bae,
N.J.Reiter,
C.A.Bingman,
S.S.Kwan,
D.Lee,
G.N.Phillips,
S.E.Butcher,
D.A.Brow.
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Ref.
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J Mol Biol, 2007,
367,
1447-1458.
[DOI no: ]
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PubMed id
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Abstract
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The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24
(Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA
base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of
the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6
bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The
interaction partners of each of the four RRMs of Prp24 and how these
interactions direct U4/U6 pairing are not known. Here we report the crystal
structure of the first three RRMs and the solution structure of the first two
RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs
1 and 3. These contacts occupy much of the canonical RNA-binding faces
(beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed.
Previously identified substitutions in Prp24 that suppress mutations in U4 and
U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a
conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate
that a large basic patch evident on the surface of RRMs 1 and 2 is part of a
high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6
RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure.
The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with
an unidentified ligand.
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Figure 4.
Figure 4. Solution structure of Prp24-12. (a) Ensemble of ten
energy-minimized NMR structures of Prp24-12. The backbone RMSD
of secondary structure-containing regions of RRMs 1 (yellow) and
2 (orange) is 0.97(±0.09) Å. (b) Ribbon diagram of
the lowest free energy structure of Prp24-12, with RRMs colored
as in Figure 1, Figure 2 and Figure 3. (c) Comparison of the
mean NMR and crystal (depicted in grey) structures. Backbone
atoms of secondary structure regions were fit to RRM domains 1
and 2 and give an RMSD of 2.0(±0.3) Å
(Supplementary Table III).
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Figure 8.
Figure 8. Mapping of U6 RNA binding interactions on the
surface of Prp24. (a) Region of the ^1H-^15N HSQC-TROSY spectra
showing amide peaks of 0.2 mM ^2H,^15N-labeled Prp24-N12 protein
in the absence (black) and presence (red) of 50 μM
21-nucleotide RNA (yeast U6 nucleotides 40–60). Assigned
resonances that are selectively line broadened upon the addition
of RNA are circled in magenta and labeled. (b) Location of the
line broadened amino acid residues (shown in magenta) in the
crystal structure. The structure at left is in the same
orientation as is shown in Figure 3(a) and (b), and is rotated
180° around the vertical axis relative to the structure at
right. The ribbon is colored as in Figure 1. (c) Electrostatic
potential isosurface of the Prp24-N123 crystal structure
calculated by the Poisson-Boltzmann equation. The isosurface is
contoured at ±4 kT and is superposed on the molecular
surface (white). A prominent electropositive region (blue)
exists on one side of the protein, possibly corresponding to an
RNA binding site. Electronegative regions (red) are small and
distributed more uniformly around the protein. Structures are in
the same orientation as in (b).
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
367,
1447-1458)
copyright 2007.
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