Biochemical and Physiological Properties of the DNA
Binding Domain of AraC Protein
Andrew Timmes, Michael Rodgers and Robert Schleif*
Department of Biology, Johns
Hopkins University, 3400
N. Charles St., Baltimore
MD 21204, USA
Intact AraC protein is poorly soluble and difficult to purify, whereas its
dimerization domain is the opposite. Unexpectedly, the DNA binding
domain of AraC proved also to be soluble in cells when overproduced
and is easily purified to homogeneity. The DNA binding affinity of the
DNA binding domain for its binding site could not be measured by
electrophoretic mobility shift because of its rapid association and dis-
sociation rates, but its affinity could be measured with a fluorescence
assay and was found to have a dissociation constant of 1 £ 1028M in
100 mM KCl. The binding of monomers of the DNA binding domain to
adjacent half-sites occurs without substantial positive or negative coopera-
tivity. A simple analysis relates the DNA binding affinities of monomers
of DNA binding domain and normal dimeric AraC protein.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: DNA binding domain; monomer affinity; cooperativity;
induction mechanism; AraC
Many proteins whose binding to DNA is modu-
lated by ligands are oligomers, likely because their
DNA binding affinity will be high when two
domains can be properly positioned for binding to
two DNA sites, and low when either the presence
or absence of ligand leads to improper positioning
of the domains.1Such a regulation mechanism
requires that the DNA binding affinity of a single
subunit or a single DNA binding domain of such
a protein be too low to generate an appreciable bio-
logical response. Similarly, proteins whose DNA
binding is controlled by oligomerization must
bind DNA weakly as monomers and more tightly
AraC protein from Escherichia coli, the dimeric
regulator of the L-arabinose operon, is a ligand
responsive protein that changes its DNA binding
affinity in response to arabinose.1The DNA bind-
ing domain of AraC, when in monomeric form,
has a lower affinity for DNA than the dimer and
leads to negligible induction of the araBADoperon
in AraC2cells.2When artificially dimerized by a
leucine zipper2or a covalent linkage,3however,
the DNA binding domain is able to fully induce
the operon, showing that the domain contains
The issue of increasing the DNA binding affinity
of a protein by dimerizing it or, more generally, of
modulating a protein’s DNA binding affinity by
controlling the relative positioning of its DNA
binding domains, raises the general question of
what relationship is expected between the DNA
binding affinity of monomers and dimers of a
protein. On one hand, if two binding units such as
the two parts of desthiobiotin, 4-methylimidazoli-
done and hexanoate, are held rigidly in the same
relative positions as they must occupy when
bound to a receptor, the binding affinity and
apparent binding cooperativity between the two
5 £ 1025M and 3.5 £ 1024M from streptavidin, but
1.2 £ 10212M.4On the other hand, if the two units
are connected by a flexible linker, the binding of
one unit becomes independent of the other and no
cooperativity is exhibited as the linker becomes
very long. What about intermediate cases with
relatively short and flexible linkers? In special
cases, it may be possible to measure the binding
affinity of both monomers of a DNA binding
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
E-mail address of the corresponding author:
Abbreviations used: DBD, DNA binding domain; BSA,
bovine serum albumin.
doi:10.1016/j.jmb.2004.05.018J. Mol. Biol. (2004) 340, 731–738
This workwas supportedbyNIH grant
GM18277 (to R.S.).
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Edited by M. Yaniv
(Received 27 February 2004; received in revised form 29 April 2004; accepted 25 May 2004)
AraC DNA Binding Domain