From the Biology Department, Johns Hopkins University, Baltimore, Maryland 21218
Received for publication, September 22, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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Constitutive mutations were sought and found in
the N-terminal arm of the Escherichia coli regulatory
protein of the arabinose operon, AraC protein. A new mutation, N16D,
was of particular interest. Asn-16 is not seen in the crystal
structure of the AraC dimerization domain determined in the absence of
arabinose, because the N-terminal arm 18 residues are disordered, but
in the presence of arabinose, residues 7-18 fold over the arabinose
and make many interactions with it. In this state Asn-16 lies near two
positively charged amino acids, Lys-43 and Arg-99. We propose that the
introduction of the negatively charged aspartic residue at position 16 creates a charge-charge interaction network among Asp-16, Lys-43, and Arg-99 that holds the arm to the dimerization domain even in the absence of arabinose. This frees the DNA-binding domains and allows them to bind cis to
I1-I2 half-sites and activate
transcription. Mutating the two positively charged residues to alanines
individually and collectively decreased or eliminated the
constitutivity created by the N16D mutation.
The regulatory protein of the L-arabinose operon in
Escherichia coli is thought to function by what is called
the light switch mechanism (1, 2). In the absence of arabinose, the two
DNA-binding domains of the dimeric AraC molecule are held not only by
the eight amino acid peptide linkers between the dimerization domain and the DNA-binding domain but also by the N-terminal arms from the
dimerization domains that bind to the DNA-binding domains. Even though
each of these two connections may be flexible (3), their combination
restricts the orientations freely available to the DNA-binding domains.
This restriction makes it energetically disadvantageous for the two
domains to contact the adjacent I1 and
I2 half-sites but rather easy for the two
domains to contact the nonadjacent half-sites I1
and O2 and form a DNA loop, represented in Fig.
1 (4). The situation changes upon the
binding of arabinose. The binding of arabinose in the dimerization
domains favors binding of the arms to the dimerization domains rather
than the DNA-binding domains. This relieves the constraints on the
DNA-binding domains and allows them to bind to the adjacent direct
repeat I1 and I2 half-sites. This mode of DNA binding allows AraC to activate RNA polymerase for transcription from pBAD
(5-9).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The light switch model of the response of
AraC to arabinose.
According to the light switch mechanism, the binding of arabinose to
the dimerization domain is normally required for the N-terminal arms to
prefer to bind to the dimerization domains rather than the DNA-binding
domains (1, 2). This model predicts the existence of two kinds of
mutations in AraC that would allow it to activate transcription in the
absence of arabinose. In the first type, the interactions between the
arms and DNA-binding domains are weakened by mutations in either the
arm or domain. In the second type, the arms are kept from the
DNA-binding domains as a result of strengthened interactions between
the arms and the dimerization domains that lead the arm to bind to the
dimerization domain even in the absence of arabinose. Because either
type of mutation frees the DNA-binding domains in the absence of
arabinose, AraC activates the pBAD promoter in
the absence of arabinose, that is, it becomes constitutive. Although a
sizeable number of constitutive mutations in AraC have been isolated
(10-12), no mutations of the second type have been identified.
Although it is quite possible that among the known constitutive
mutations, some do have the effect of strengthening the
arm-dimerization domain interaction, none stands out as obviously being
of this type as judged from simple inspection of the structure of the
dimerization domain that has been determined in the presence of
arabinose (13). We have therefore sought additional constitutive
mutations in AraC in hopes of finding mutations possessing a clear
mechanistic basis and that would either support or refute the light
switch mechanism.
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EXPERIMENTAL PROCEDURES |
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Isolation of Constitutive Mutations--
For mutagenesis,
plasmid pWR03 (2) containing the entire AraC coding region was
transformed into mutator cells (endA1 gyrA96 thi-1 hsdR17 supE44
relA1 lac mutD5 mutS mutT Tn10:Tetr) (E. coli XL1-Red competent cells; Stratagene, La Jolla, CA) as
described by Stratagene. The mutagenized plasmids were harvested via
resin-based mini-prep (Wizard Plus DNA Miniprep purification system;
Promega) and transformed into SH321 (F
araC-leu1022
lac74
galK
Strr thi1)
(14). Constitutive mutant candidates were selected based on their
abilities to grow on minimal arabinose-fucose plates containing 0.1%
arabinose and 0.2% fucose. Colonies that grew up in 2 days were
further tested by in vivo arabinose isomerase assay.
Arabinose Isomerase Assay-- Cells were grown in M10 minimal salts medium plus 0.4% glycerol, 10 µg/ml thiamine, 0.02% L-leucine, and 0.2% casamino acids (15). When cells reached an A600 of ~0.8, they were harvested and assayed for their arabinose isomerase activities as described (15). When induction by arabinose was necessary, 0.2% arabinose was added to the culture 45 min before the assay.
Site-directed Mutagenesis--
Site-directed mutagenesis was
carried out using the Stratagene QuickChangeTM
site-directed mutagenesis kit (Stratagene, La Jolla, CA). The following
reagents were mixed together: 125 ng each of the two primers, 50 ng of
parental plasmid template, 1 µl of 10 mM dNTP mix, 5 µl
of 10× reaction buffer, 1 µl of Pfu DNA polymerase (2.5 units/µl; Stratagene, La Jolla, CA), and dH2O to a final
volume of 50 µl. All the other steps were as described by the
Stratagene manual.
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RESULTS |
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Isolation of Ara Constitutive Mutants--
Induction of the
arabinose operon is inhibited by the L-arabinose analogue,
D-fucose (10, 12), but mutants can be isolated that are
resistant to this inhibition by selecting for growth on medium where
the only carbon sources are arabinose and fucose. Such mutants are
frequently found to be constitutive (10, 16). To isolate constitutive
mutants, we therefore passed a plasmid coding for the AraC gene through
a mutator E. coli strain XL1-Red, which is deficient
in three of the primary DNA repair pathways. After accumulating
mutations during propagation in the mutator cells, the plasmid was
transformed into an AraC strain.
Constitutive mutants, present at a frequency of ~0.2% among the transformants, were selected on the basis of their abilities to grow on minimal arabinose-fucose plates. The arabinose isomerase activities of the candidates were then assayed after growth in minimal medium in the absence of arabinose, and those with isomerase levels at least 3-fold above the wild type basal level were sequenced. Four different mutations were found, all lying in the N-terminal arm of AraC. They were L9K, S14P, N16D, and L19P. Of these, L19P was isolated twice, and N16D was isolated three times; L9K and S14P were isolated only once.
Constitutive mutations at positions 9, 14, and 19 have been isolated before (1, 12). The mutation N16D was new, however. Arabinose isomerase assay showed that, in the absence of arabinose, the N16D mutant activates the pBAD promoter 67%, as well as wild type AraC induces in the presence of arabinose. By comparison, wild type AraC induces the pBAD promoter less than 1% of the full induction level in the absence of arabinose.
In the structure of the AraC dimerization domain determined in the
absence of arabinose, residues 1-18 are not seen, because the arms are
disordered. In the structure determined in the presence of arabinose,
however, the arms starting from residue 7 are visible and are seen to
fold over the bound sugar. Asn-16 is close to two positively charged
amino acids, Lys-43 and Arg-99 (Fig. 2). The closest distances between the side chains of the three residues are
as follows: Asn-16Lys-43, 3.5 Å; Asn-16
Arg-99, 2.0 Å; and Lys-43
Arg-99, 5.6 Å. It is very likely that changing residue 16 from
asparagine to negatively charged aspartic acid creates a charge-charge
interaction network between it and Lys-43 and Arg-99. We expect that,
with the additional binding energy provided by this interaction, the
arms are held to the dimerization domains most of the time even in the
absence of arabinose, and this leads the protein to behave as though
arabinose were present.
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Effects of Alanine Substitutions Lys-43 and Arg-99-- The hypothesis that in the N16D mutant the arms are held to dimerization domains through charge-charge interactions was tested by mutating the two positively charged amino acids Lys-43 and Arg-99 to alanines individually and collectively. If the electrostatic interactions increasing the strength of the binding of the arm to the core of the dimerization domain cause AraC to behave constitutively, then the phenotypes of the double or triple mutants should change accordingly. Alanine substitutions of Lys-43 and Arg-99 were generated by site-directed mutagenesis. The abilities of the mutants to activate the pBAD promoter were examined by the arabinose isomerase assay both in the absence and in the presence of arabinose. Table I shows that changing either Lys-43 or Arg-99 to alanine in N16D AraC resulted in a partial loss of the constitutivity and that when both were changed to alanines, AraC no longer displayed any constitutive behavior. The triple mutant, N16D,K43A,R99A, behaved almost the same as the wild type AraC. As shown in Table I, the alanine substitutions did not affect the activation ability of either the wild type AraC or N16D in the presence of arabinose. The results are consistent with the idea that mutation N16D creates stronger arm-dimerization domain interactions and that these make AraC constitutive.
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DISCUSSION |
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We searched for and found constitutive mutations in AraC protein. One of the mutations found, N16D, was not only new, but its mechanism of action can be understood and is consistent with the light switch mechanism for AraC action. In the three-dimensional structure of the AraC dimerization domain, Asn-16 lies very close to the two positively charged residues Lys-43 and Arg-99. Presumably then, a salt-bridge triad arises among Asp-16, Lys-43, and Arg-99, and the extra energy provided by this electrostatic network holds the arms to the dimerization domain even in the absence of arabinose.
Studies by both x-ray crystallography and NMR show that in almost all cases, the structural changes generated by changing the charge of a surface-exposed amino acid residue are confined to the immediate vicinity of the mutation (17-19). We expect this to be true of N16D, as well, although our attempts to crystallize the dimerization domain bearing the N16D mutation yielded only a crystal form with a space group that is unsuitable for structural determination. Under similar conditions wild type AraC dimerization domain also prefers to form these same unsuitable crystals but also occasionally forms crystallographically useful crystals (13).
We think it likely that the salt bridges formed in the N16D mutant
significantly stabilize the arm-domain interactions. Although some
report that single salt bridges on the surfaces of protein contribute
little stability (17-21), it is worth pointing out that some of the
cited studies involved engineered ion pairs that may not have been in
configurations necessary for optimal salt bridges. Moreover, when salt
bridges involve three charged amino acids rather than two,
cooperativity ought to add to the stabilizing effects (17). For
example, on the surface of barnase are two exposed salt bridges between
the C-terminal Arg-110 and aspartic residues at positions 8 and 12 in
the first helix of the protein. The contribution to the stability
of the protein by the salt bridge between Asp-12 and Arg-110 is
1.25 kcal/mol, whereas that of the salt bridge between Asp-8 and
Arg-110 is 0.98 kcal/mol. The energy of each is reduced by 0.77 kcal/mol when the other is absent, indicating that the two salt bridges
are coupled.
Each of the two residues, Lys-43 and Arg-99, contributes to the
constitutivity of the N16D mutant as shown by the fact that changing
either of the two positively charged residues to alanine substantially
reduced the level of constitutivity. Thus, a triad similar to the one
found in barnase could form, and coupling between the two salt bridges
would take place. The resulting stabilizing force is large enough for
the arms to prefer binding to the core of the dimerization domain
rather than to the DNA-binding domain or not binding to either domain.
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FOOTNOTES |
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* 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: Biology Dept., 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, November 7, 2000, DOI 10.1074/jbc.M008705200
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