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Title
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Adjacent zinc-finger motifs in multiple zinc-finger peptides from SWI5 form structurally independent, flexibly linked domains.
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Authors
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Y.Nakaseko,
D.Neuhaus,
A.Klug,
D.Rhodes.
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Ref.
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J Mol Biol, 1992,
228,
619-636.
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PubMed id
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Abstract
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Peptides containing either one, two or three of the three zinc-finger motifs
from the yeast transcription factor SWI5 have been prepared by expression in
Escherichia coli. The DNA binding characteristics of these peptides were
investigated, and a two-dimensional nuclear magnetic resonance (n.m.r.) study
undertaken to establish the three-dimensional structures of the two-finger
peptide. The peptide containing fingers 1 and 2 binds sequence specifically to
two thirds of the DNA binding site recognized either by intact SWI5 or by the
isolated three-finger peptide, and hence has the correct tertiary fold for DNA
recognition. These results also establish the polarity of DNA binding, since the
N-terminal two fingers of SWI5 bind to the 5' end of the DNA binding site. Mild
proteolysis of the three-finger peptide using trypsin results in a small number
of discrete products, which is consistent with the presence of three structured
mini-domains. Nearly complete n.m.r. signal assignments were obtained for two
peptides containing finger 2 alone or fingers 1 + 2. Comparison of
two-dimensional spectra of these peptides and others clearly shows that the NOE
enhancements and chemical shifts characteristic of each finger are quite
insensitive to the presence or absence of neighbouring fingers. This clearly
indicates that adjacent zinc-finger domains are structurally independent in
these peptides from SWI5. However, there must be some steric limitations on the
possible relative orientations of the fingers, and to establish limits for these
a set of structures for the peptide containing fingers 1 + 2 was calculated
using the YASAP simulated annealing protocol in conjunction with n.m.r.-based
constraints. A more detailed description of the three-dimensional structures of
finger 1 and finger 2, and their relationship to other previously determined
structures of single zinc-fingers, is given in the accompanying paper.
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