The C-terminal End of AraC Tightly Binds to the Rest of Its
Domain*
Tara L.
Harmer and
Robert
Schleif
From the Department of Biology, Johns Hopkins University,
Baltimore, Maryland 21218
Received for publication, August 30, 2000, and in revised form, November 13, 2000
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ABSTRACT |
Genes were synthesized to express two DNA binding
domains of AraC connected by short linkers. The abilities of the
resulting proteins to bind to DNA containing AraC half-sites separated
by the usual four bases as well as an additional two or three helical turns of the DNA were measured. The inability of some of the protein constructs to bind to widely separated half-sites indicates that the
C-terminal 14 amino acids of AraC are firmly bound to the rest of the
DNA binding domain.
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INTRODUCTION |
More than 100 different proteins currently can be found in
sequence data bases to possess significant sequence identity and a
similarity to the DNA binding domain of AraC, the regulator of the
arabinose operon in Escherichia coli. The pairwise identity between these domains often is below the 25% threshold for assurance that a pair of proteins possesses the same tertiary structure (1, 2).
Nonetheless, the fact that the family of homologs is so large, and the
fact that many whose functions have been determined are transcriptional
regulators and that similarity extends across 120 amino acids all
greatly raise the likelihood that the family members have the same
structure. The structures of two members, MarA (3) and the DNA
binding domain of Rob (4), with a 40% identity between the two and 16 and 20% identity, respectively, to the DNA binding domain of AraC have
been determined and do have the same structure.
AraC has a poor similarity with MarA and Rob over its final 14 amino
acids residues 282-292 (Fig. 1). Is this
region a structureless tail extending into the solvent, or is it firmly
bound to the protein as is the same sequence region of MarA and Rob?
This information is important in the construction of variants of AraC
in which arms must be added to the protein to enable it to bind to
other proteins. The engineering of such arm domain interactions is
appealing in the construction of alternative regulation schemes
(5).

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Fig. 1.
AraC DNA binding domain MarA and Rob sequence
alignment. Alignment performed using ClustalW 1.8 with a gap
extension penalty of 3 is shown (14).
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As of yet, no structure has been reported for the DNA binding domain of
AraC. Thus, structure information about the DNA binding domain of AraC
and particularly whether the C-terminal amino acids of the protein are
free in the solvent or bound to the rest of the domain must be obtained
by indirect means. By connecting two DNA binding domains of AraC with
arms connecting to different places on the DNA binding domain, we have
been able to show that the final 14 amino acids are firmly bound to the
rest of the domain.
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EXPERIMENTAL PROCEDURES |
Genes coding for the double DNA-binding proteins and DNA used in
the DNA migration retardation assays were synthesized by polymerase
chain reaction using oligonucleotides approximately 60 nucleotides long, purchased from Integrated DNA Technologies, synthesized at the 200 nM scale, purified by polyacrylamide
gel electrophoresis, and dissolved in dH2O at 1 mg/ml.
The sequences of the
I1I2,
I1-21-I1, and
I1-32-I1 DNAs used
in the migration retardation assay were
TTTGCTAGCCCTAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACATATCGCGTTTACT, TTTGCTAGCCCTAGCATTTTTATCCATAGCTACTGGTACCGTCTCATGGAAATTAGCATTTTTATCCATATCGCGTTTACT, and
TTTGCTAGCCCTAGCATTTTTATCCATAGCCTAGCGTCGTTGACTGGTACGGTCTCACAG AGATTAGCATTTTTATCCATATCGCGTTTACT, where the
I1 or I2 half-sites are underlined.
The double DNA-binding proteins were constructed using the two-step
polymerase chain reaction protocol as described previously (6). All
genes described here were cloned into pSE380, a 4.4-kilobase plasmid
containing the promoter ptrc, a
ColE1 origin of replication, lacO operator,
LacIq repressor gene, and ampicillin
resistance (Invitrogen, San Diego, CA). Plasmid constructs are
summarized in Table I.
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Table I
Plasmids constructed
The connector shows the final amino acid of the first AraC DNA binding
domain, the connector sequence including any natural AraC linker, and
the first amino acid of the second AraC DNA binding domain.
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The first DNA binding domain of each double domain protein consists of
AraC amino acids 169-283 at least, and the second DNA binding domain
consists of AraC amino acids of at least 175-292. For the linkers
connecting the DNA binding domains, we chose amino acids from the
linker region of the yeast mating-type repressor protein,
2 (7). The
linker provides the yeast repressor protein with sufficient flexibility
that it can bind to a variety of spacings and orientations of its DNA
half-sites (8). The linker was flanked on either side by two amino
acids dictated by the restriction endonuclease sites EcoRI
and BamHI. The proteins were expressed in strain SH321
(F
, araC
leu1022,
lac74, galK
, strr) (9).
Arabinose isomerase was assayed as described (10). Cells were grown in
liquid minimal salts media with 0.4% glycerol and 0.4% casamino acids
to an apparent optical density at A550 nm of
between 0.300 and 0.600 in the presence and absence of 0.2% arabinose
as noted.
DNA migration retardation assays were performed as described
(11). Proteins were overexpressed in a 5-ml culture, which had been
inoculated with 16 µl of stationary phase SH321 cells and then grown
in the absence followed by the presence of
isopropyl-1-thio-
-D-galactopyranoside for 90 min.
Cells were pelleted by centrifugation, resuspended in 300-µl buffer
containing 100 mM K2PO4, 50 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM dithioerythritol, and 1 mM phenylmethyl
sulfonylfluoride, and then lysed by sonication. Samples were
centrifuged, and the supernatant was added to 175 µl of 100%
glycerol. Western transfer experiments coupled with stained SDS gels
show that the various linker proteins constitute about one-twentieth of
1% of the protein in the lysates. Radiolabeled DNA template was
incubated in the presence of calf thymus DNA, arabinose, and buffer
containing 50 mM KCl, 25 mM Na-Hepes, pH 7.4, 2.5 mM dithioerythritol, 0.1 mg/ml bovine serum albumin,
0.1 mM K-EDTA, 5% glycerol, and 1% arabinose. 1 µl of
cell lysate was added, and the reaction was incubated at 37 °C for
10 min and then loaded on a gel containing 6% acrylamide, 0.1%
bisacrylamide, 0.1% ammonium persulfate, and 0.2%
N,N,N'N'-tetramethylethylenediamine. The gel was
run in a recirculated chilled buffer containing 10 mM Tris
acetate and 1 mM K-EDTA, pH 7.4, at 75 V for 1 h. Gels
were dried under vacuum and exposed overnight to phosphor plates. A
Molecular Dynamics PhosphorImager was used to scan and analyze the gels.
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RESULTS |
The C terminus of MarA consists of amino acids 111-124,
corresponding to AraC amino acids 278-291. This C-terminal stretch lies across the side of the protein opposite the DNA binding site. If
the corresponding C-terminal stretch in AraC is attached tightly to the
core, shifting the attachment point of the linker to the first DNA
binding domain toward the C-terminal end of the protein should
effectively shorten the maximum distance that the two DNA binding
domains can be spaced when binding to direct repeat half-sites as
illustrated in Fig. 2. If instead, the
C-terminal stretch is loosely attached as also shown in Fig. 2, moving
the attachment point toward the C terminus of the protein effectively
lengthens the linker and increases the maximum distance that the two
DNA binding domains can be separated when binding in a direct repeat orientation.

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Fig. 2.
Two AraC DNA binding domain protein linkers
attached to different points on the first DNA binding domain. If
the C-terminal stretch is tightly bound as shown in the top
diagram, the effect is shortening, but if the C-terminal stretch
is not bound, the effect is lengthening as shown in the bottom
diagram.
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To test whether the C-terminal stretch of AraC is held firmly against
the body of the DNA binding domain or whether it is loosely attached,
we performed a series of DNA binding assays with the double AraC DNA
binding domain proteins and three DNA templates, wild type ara
I1I2,
I1-21-I1, which contains
two helical turns between the half-sites, and
I1-32-I1, which contains
three helical turns. If the C-terminal stretch is held tightly to the body, shifting the linker attachment point to the first DNA binding domain from AraC amino acids 263 to 292 will result in a shortening of
the maximum distance of separation between the centers of the structured cores of the two DNA binding domains and decrease the binding ability to widely spaced DNA binding sites. However, if the
association is weak, shifting the linker attachment point will result
in a lengthening of the maximum separation among the centers of the
structured cores of the DNA binding domains. This will allow the
protein to bind to DNA with more widely separated half-sites. Fig.
3 and Table
II show that pTH11 binds not as well as
pTH13 does with increasing distance between the binding sites. This
result indicates that the C-terminal stretch, indeed, is held firmly to
the body of the protein.

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Fig. 3.
Binding of AraC and AraC derivative
DNA binding domain proteins to three DNA templates,
I1I2
,
I1-21-I1, and
I1-32-I1.
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Table II
Summary of results
Linker length is the number of amino acids between attachment points to
the two AraC DNA binding domains. Attachment point indicates the AraC
amino acid position where the linker attaches to the first DNA binding
domain. The maximum reach is the maximum distance between the centers
of the structured cores of the two DNA binding domains when binding to
half-sites in a direct repeat orientation when the C-terminal 14 amino
acids are strongly bound to the body of the DNA binding domain.
Induction at
I1I2pBAD was
measured by arabinose isomerase assay, and the results are reported as
the percent of wild-type AraC activity. Binding to template DNA was
determined by in vitro DNA migration retardation assays, and
the number reported is the fraction of DNA found in the protein-DNA
complex.
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DISCUSSION |
In this work, we have shown that the C terminus of AraC residues
278-292 binds tightly to the body of the AraC DNA binding domain. This
was determined by changing the attachment points of the linker to the
first DNA binding domain of double AraC DNA binding domain proteins,
assaying the binding abilities of the resulting proteins to DNA
templates with AraC half-sites separated by the wild-type spacing, and
assaying the binding abilities of the resulting proteins to DNA
templates with the half-sites separated by two and three additional
helical turns of the DNA. The binding of the C-terminal stretch
to the body of AraC is strong enough that this interaction remains
intact when attached to a second AraC DNA binding domain via a flexible
linker and when this entire protein is bound to DNA. The binding
energies required to bind a double AraC DNA binding domain protein to
I1 half-sites separated by three helical turns are
insufficient to strip the C-terminal stretch from the body of the DNA
binding domain.
This work also provides data against a proposal for the mode of
dimerization of AraC in the absence of arabinose and the mechanism of
response to arabinose made on the basis of the crystal packing arrangements of the dimerization domain of AraC in the presence and
absence of arabinose (12). AraC protein was proposed to loop between
distal DNA half-sites and repress the araBAD operon in the
absence of arabinose when dimerized by a face to face interaction that
allowed the DNA binding domains to be separated by 140 Å or greater
and to cease looping and activate transcription when the separation
distance was decreased by a shift in the dimerization interface. The
proteins encoded by pTH15 and pTH16 allow greater than 140-Å
separations, and as shown in Table II, strongly induce the ara
pBAD promoter. Other data also show that the alternative interface mechanism is inapplicable to AraC (13).
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FOOTNOTES |
*
This work supported by National Institutes of Health Grant
GM18277.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biology,
Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel.: 410-516-5206; Fax: 410-516-5213; E-mail:
bob@gene.bio.jhu.edu.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M007956200
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REFERENCES |
1.
|
Sander, C.,
and Schneider, R.
(1991)
Proteins
9,
56-68[Medline]
[Order article via Infotrieve]
|
2.
|
Holm, L.,
Ouzounis, C.,
Sander, C.,
Tuparev, G.,
and Vriend, G.
(1992)
Protein Sci.
1,
1691-1698[Abstract/Free Full Text]
|
3.
|
Rhee, S.,
Martin, R. G.,
Rosner, J. L.,
and Davies, D. R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10413-10418[Abstract/Free Full Text]
|
4.
|
Kwon, H. J.,
Bennik, M. H.,
Demple, B.,
and Ellenberger, T.
(2000)
Nat. Struct. Biol.
7,
424-430[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Schleif, R.
(1999)
Proteins
34,
1-3[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Prytulla, S.,
Dyson, J. H.,
and Wright, P. E.
(1996)
FEBS Lett.
399,
283-289[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Astell, C.,
Ahlstrom-Jonasson, R.,
and Smith, M.
(1981)
Cell
27,
15-23[Medline]
[Order article via Infotrieve]
|
8.
|
Sauer, R. T.,
Smith, D. L.,
and Johnson, A. D.
(1988)
Genes Dev.
2,
807-816[Abstract]
|
9.
|
Hahn, D.,
Dunn, T.,
and Schleif, R. F.
(1984)
J. Mol. Biol.
180,
61-72[Medline]
[Order article via Infotrieve]
|
10.
|
Schleif, R. F.,
and Wensink, P.
(1981)
Practical Methods in Molecular Biology
, Springer-Verlag New York Inc., New York
|
11.
|
Hendrickson, W.,
and Schleif, R.
(1984)
J. Mol. Biol.
174,
611-628
|
12.
|
Soisson, S. M.,
MacDougall-Shackleton, B.,
Schleif, R.,
and Wolberger, C.
(1997)
Science
276,
421-425[Abstract/Free Full Text]
|
13.
|
Saviola, B.,
Seabold, R.,
and Schleif, R. F.
(1998)
J. Mol. Biol.
278,
539-548[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Thompson, J. D.,
Higgins, D. G,
and Gibson, T. J.
(1994)
Nucleic Acids Res.
22,
4673-4680[Abstract]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.