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PDBsum entry 2vrd
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Nuclear protein
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PDB id
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2vrd
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References listed in PDB file
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Key reference
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Title
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The structure and biochemical properties of the human spliceosomal protein u1c.
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Authors
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Y.Muto,
D.Pomeranz krummel,
C.Oubridge,
H.Hernandez,
C.V.Robinson,
D.Neuhaus,
K.Nagai.
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Ref.
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J Mol Biol, 2004,
341,
185-198.
[DOI no: ]
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PubMed id
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Abstract
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The spliceosomal U1C protein is critical to the initiation and regulation of
precursor messenger RNA (pre-mRNA) splicing, as part of the U1 small nuclear
ribonucleoprotein particle (snRNP). We have produced full-length and 61 residue
constructs of human U1C in soluble form in Escherichia coli. Atomic absorption
spectroscopy and mass spectrometry show that both constructs contain one Zn atom
and are monomeric. Gelmobility-shift assays showed that one molecule of
recombinant U1C, either full-length or 61 residue construct, can be incorporated
into the U1 snRNP core domain in the presence of U1 70k. This result is in
perfect agreement with the previous experiment with U1C isolated from the HeLa
U1 snRNP showing that the recombinant U1C is functionally active. We have
determined the solution structure of the N-terminal 61 residue construct of U1C
by NMR. A Cys(2)His(2)-type zinc finger, distinct from the TFIIIA-type, is
extended at its C terminus by two additional helices. The two Zn-coordinating
histidine residues are separated by a five residue loop. The conserved basic
residues in the first two helices and the intervening loop may be involved in
RNA binding. The opposite beta-sheet face with two surface-exposed Tyr residues
may be involved in protein contacts. Both the full-length and 61 residue
constructs of human U1C fail to bind RNA containing the 5' splice site sequence,
in contrast to what has been reported for the Saccharomyces cerevisiae
orthologue.
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Figure 5.
Figure 5. Solution structure of human U1C. A, Stereo ribbon
representation of the lowest-energy structure. A blue arrow
indicates the b-strands and a red coil indicates the helical
region. Some aromatic residues including the two tyrosine
residues (Y5 and Y12) on the b-strands as well as the
Zn-coordinating residues (Cys6, Cys9, His24 and His30) are
indicated. B, The surface representation of U1C with surface
electrostatic potential. The surface formed by the three
a-helices displays many positively charged amino acid residues.
Blue, positively charged; red, negatively charged. C, The
b-strand surface rich in aromatic and negatively charged amino
acid residues. Figures were drawn using MOLSCRIPT[53.] and
GRASP. [54.]
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Figure 6.
Figure 6. Filter-binding assay of the 5' splice site
binding to U1 snRNP and U1C protein. ( o ) The reconstituted U1
snRNP; (sB) full-length U1C. The experiments were done in
triplicate and error bars are shown for U1 snRNP only for
clarity.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2004,
341,
185-198)
copyright 2004.
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