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PDBsum entry 1p47
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Transcription/DNA
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PDB id
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1p47
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Constraints for zinc finger linker design as inferred from X-Ray crystal structure of tandem zif268-Dna complexes.
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Authors
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E.Peisach,
C.O.Pabo.
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Ref.
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J Mol Biol, 2003,
330,
1-7.
[DOI no: ]
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PubMed id
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Abstract
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Zinc-finger proteins offer a versatile and effective framework for the
recognition of DNA binding sites. By connecting multiple fingers together with
canonical TGEKP linkers, a protein may be designed to recognize almost any
desired target DNA sequence. However, proteins containing more than three
zinc-fingers do not bind as tightly as one might predict, and it appears that
some type of strain is introduced when a six-finger protein is constructed with
canonical linkers. In an attempt to understand the sources of this strain, we
have solved the 2.2A resolution X-ray crystallographic structure of a complex
that has two copies of the three-finger Zif268 protein bound to adjacent sites
on one duplex DNA. Conceptually, this is equivalent to a six-finger protein in
which the central linker has been removed and the complex has been allowed to
"relax" to its most stable conformation. As in other Zif268-DNA
complexes, the DNA is approximately linear and is slightly underwound.
Surprisingly, the structure of the complex is similar (within 0.5A) to an
arrangement that would allow a canonical linker at the center of the complex,
and it seems possible that entropic effects (involving the librational degrees
of freedom in the complex) could be important in determining optimal linker
length.
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Figure 2.
Figure 2. Model of two Zif268 molecules bound to tandem
binding sites. Fingers 1-3 are bound right to left. Fingers 1, 2
and 3 are colored in magenta, yellow, and orange, respectively.
The red line indicates the gap for which a linker would be
designed.
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Figure 3.
Figure 3. Alignment of the first finger of Zif268 protein
from the 1.6 Å 1AAY structure with the third finger of
molecule A. The 1AAY structure is in cyan. The DNA in this
structure is represented in green and only the region from Thy7
to Thy16 of the primary strand is shown. Finger 3 of molecule A
is in orange, the first and second fingers of molecule B are in
magenta and yellow.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2003,
330,
1-7)
copyright 2003.
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Secondary reference #1
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Title
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Zif268 protein-Dna complex refined at 1.6a: implications for understanding zinc finger DNA recognition
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Authors
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M.Elrod-Erickson,
M.A.Rould,
L.Nekludova,
C.O.Pabo.
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Ref.
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structure, 1996,
6,
451.
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Secondary reference #2
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Title
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Getting a handhold on DNA: design of poly-Zinc finger proteins with femtomolar dissociation constants.
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Authors
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J.S.Kim,
C.O.Pabo.
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Ref.
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Proc Natl Acad Sci U S A, 1998,
95,
2812-2817.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Structure-based design of a six-finger peptide,
268//NRE. The cocrystal structure of the Zif268-DNA complex (16,
19) and^ the template B-DNA (used at the junction) were aligned
by superimposing phosphates. In this model, two three-finger
peptides bind to corresponding 9-bp sites (bases shown in white)
separated by a 2-bp gap (bases shown in gray). Note that the
orientation of one three-finger peptide almost exactly matches
that of the other three-finger peptide because one helical turn
of this underwound DNA contains 11 bp.
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Figure 2.
Fig. 2. Schematic representations of zinc finger peptides
and of reporter constructs used in transfection studies. (A)
Zinc finger peptides. Each finger is represented by a circle.
The amino acid^ sequence of a linker in the Zif268 peptide
(which has a canonical "TGEKP" linker) is shown, and longer
linkers used to connect the^ three-finger peptides are indicated
below. In each case, the box on the left denotes the helical
region and includes the second^ of the conserved His residues of
the finger; the zigzag line denotes the first  -sheet of
the next finger, which includes the first of the conserved Cys
residues. (B) Promoters of luciferase reporter genes. The
nucleotide positions of the TATA box, the start codon, and zinc
finger binding sites are numbered with respect to the^
transcription start site (+1).
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