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PDBsum entry 2vi6
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Transcription
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PDB id
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2vi6
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References listed in PDB file
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Key reference
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Title
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Crystal structure and DNA binding of the homeodomain of the stem cell transcription factor nanog.
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Authors
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R.Jauch,
C.K.Ng,
K.S.Saikatendu,
R.C.Stevens,
P.R.Kolatkar.
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Ref.
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J Mol Biol, 2008,
376,
758-770.
[DOI no: ]
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PubMed id
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Abstract
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The transcription factor Nanog is an upstream regulator in early mammalian
development and a key determinant of pluripotency in embryonic stem cells. Nanog
binds to promoter elements of hundreds of target genes and regulates their
expression by an as yet unknown mechanism. Here, we report the crystal structure
of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA
element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among
HD sequences, appear to affect the mechanism of nonspecific DNA recognition as
well as maintain the integrity of the structural scaffold. To assess selective
DNA recognition by Nanog, we performed electrophoretic mobility shift assays
using a panel of modified DNA binding sites and found that Nanog HD
preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis
experiments probing the role of six variant residues of Nanog on its DNA binding
function establish their role in affecting binding affinity but not binding
specificity. Together, the structural and functional evidence establish Nanog as
a distant member of a Q50-type HD despite having considerable variation at the
sequence level.
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Figure 3.
Fig. 3. DNA contact interface. Recognition helix of mNanHD
(yellow) and Msx1 (blue) and the DNA from the Msx1–DNA complex
structure are shown in the cartoon depiction. Side chains of
residues found at positions K43(mNanHD)/T43(Msx1),
T47(mNanHD)/I47(Msx1), Q50, N51, and M54(mNanHD)/A54 (Msx1) and
nucleotides of the T[1]A[2]A[3]T[4]T[5]G[6] motif as found in
the Msx1 structure are shown in the ball-and-stick
representation (PDB ID 1ig7).
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Figure 4.
Fig. 4. Binding affinity. (a) EMSAs using constructs of the
Tcf3 promoter of different lengths (30mer:
CTTTAAACCTGTTAATGGGAGCGCATTGTG; 26mer:
TTAAACCTGTTAATGGGAGCGCATTG; 22mer: AAACCTGTTAATGGGAGCGCAT;
18mer: ACCTGTTAATGGGAGCGC; 14mer: CTGTTAATGGGAGC; and 10mer:
GTTAATGGGA) at 1 nM were incubated in the presence (+) or
absence (−) of 50 nM mNanHD. The lowest band in each lane
corresponds to single-stranded DNA. (b–g) EMSA with increasing
concentrations of mNanHD (0 to 2 μM) and mutant HD constructs.
Double-stranded cy5 Tcf3 (1 nM) was used in these assays.
Representative results from at least three independent
experiments are shown. Apparent dissociation constants were
estimated by plotting the fraction of bound protein averaged
over three to five independent experiments against the total
protein concentration followed by fitting a single-site
saturation curve using SigmaPlot. The indicated error represents
the standard error of the fit.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2008,
376,
758-770)
copyright 2008.
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