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Ara Operon
Ara Operon
Keywords
allostery; transcription initiation; RNA
polymerase; solubility; fluorescence; homology;
fucose.
ATP-binding component, and AraH is the membrane-bound (Lis & Schleif, 1973). A variety of in vivo and in vitro
component. The function of an additional protein induced experiments further demonstrate the existence of catabolite
by arabinose, the araJ gene product (Reeder & Schleif, 1991), repression and that the cyclic AMP receptor protein, CAP,
is as yet unknown (Carole et al., 1999), but on the basis of its plays an important role in the induction of the ara genes
pattern of hydrophilic and hydrophobic residues, it has been (Hahn et al., 1984, 1986; Lobell & Schleif, 1991; Zhang &
conjectured to be a transporter either of arabinose-contain- Schleif, 1998; Fig. 2).
ing polymers or an exporter of arabinose. Induction of the promoters for the active transport of
arabinose, pE and pFGH, is only about 50-fold in response to
arabinose (Johnson & Schleif, 1995). The promoter for the
Expression behavior of the ara promoters synthesis of AraC, pC, also responds to the presence of
Transcription of the araBAD genes under control of the pBAD arabinose. It is also stimulated by CAP, and is repressed by
promoter is induced about 300-fold above a basal unin- the AraC protein itself both by DNA looping in which a
duced level within 3 s of the addition of arabinose to cells dimer of AraC binds to I1 and to O2, and by repression in
growing on glycerol in minimal salts medium (Schleif et al., which a dimer of AraC binds to the O1 pair of half-sites that
1973; Hirsh & Schleif, 1973; Johnson & Schleif, 1995). The partially overlap the pC polymerase-binding region. Its
induction does not require the activity of the first enzyme of regulatory behavior has been conveniently monitored by
the arabinose catabolic pathway, implying that arabinose is fusion of its promoter to the b-galactosidase gene (Casada-
the true inducer (Englesberg, 1961). This is not the case for ban, 1976). Upon arabinose addition, the promoter’s activ-
the lac operon, where b-galactosidase is required to convert ity increases up to 10-fold for about 10 min, and then reverts
lactose to the true inducer, allolactose (Bourgeois & Jobe, to almost its preinduction level (Ogden et al., 1980; Stoner &
1972). Induction of the ara genes is negligible for cells Schleif, 1983b; Hahn & Schleif, 1983).
growing on tryptone-yeast extract medium plus arabinose Because AraC is a regulatory protein, its in vivo level
until an inhibitor, probably glucose, is consumed by the cells should be very low. Only a small excess of molecules of AraC
c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved
AraC and ara regulation 781
surface as a result of the combined effects of diffusion and tionally, Lee et al. (2007) have found that the presence of
cell motility is taken up and catabolized, then rough IPTG interferes with arabinose induction and that mutants
calculations indicate that it might be reasonable to induce of AraC can be isolated in which arabinose remains an
when the extracellular arabinose concentration exceeds inducer, but the interfering ability of IPTG is much reduced.
10 7 M. The affinity of the AraC protein for arabinose, These results show that AraC also binds IPTG. That IPTG
about 0.4 mM (Ross et al., 2003), is much below this, should bind to AraC and that it might interfere with
indicating that at least one of the assumptions is far from arabinose induction is not altogether surprising. IPTG
correct. possesses a D-galactose moiety, and the ring structures of L-
On the other hand, however, the AraC protein is inside arabinose and D-galactose are identical. D-galactose itself
the cell, and the concentration of arabinose there depends appears not to be a strong inhibitor of arabinose induction
not only on the extracellular arabinose concentration but as it binds very weakly, which is consistent with Lee and
also on the rates of arabinose uptake and catabolism. colleagues finding that in addition to residues in the
c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved
AraC and ara regulation 783
of 2 109 M 1 s 1, the dissociation rate, k 1, is around binding site of AraC at the pBAD promoter is indeed a direct
0.05 s 1, and the equilibrium dissociation constant, Kd, is repeat (Carra & Schleif, 1993).
2 10 13 M (Hendrickson & Schleif, 1984). Measurement of Does a direct repeat DNA-binding site imply that the
the latter value at physiological salt concentrations required binding protein possesses a similar direct repeat subunit
special techniques to radiolabel the DNA to levels sufficient structure? If each repeat unit of the DNA were contacted by a
to allow its detection at the very low concentrations required rigid, globular subunit, then the subunits would have to
in order that the concentration of free protein in solution be possess a direct repeat relationship with respect to each
well approximated as the total protein added to the reaction other. Such a linear structure would allow additional sub-
mixture. units to bind and the formation of polymers of indefinite
At equilibrium, the rate of association of protein and length. Because AraC is a dimer in solution and was found
DNA, k1 [P] [DNA], equals the rate of dissociation, to bind DNA only as independent dimers to triple and
k 1 [PD], where k1 and k 1 are the association and quadruple direct repeat DNA half-sites, the protein must be
for induction (Schleif & Lis, 1975). Somewhat after the additional evidence for DNA looping. In addition, these
deletion mapping, it became possible to isolate plasmids showed that a dimer of AraC bound and formed the loop,
carrying the arabinose operon and to conveniently generate with one subunit binding to the upstream half-site site
and physically size deletions into the plasmid-borne ara named O2 and the other subunit binding to the I1 half-site
genes (Dunn et al., 1984; Dunn & Schleif, 1984). These (Martin et al., 1986; Lobell & Schleif, 1990).
showed that a site lying 210 bp upstream from the ara I1–I2
site was required for repression. Footprinting experiments
identified an AraC-binding site at this location.
The value of looping
Why should nature use DNA looping as a mechanism in
gene regulation? It seems to waste the DNA that constitutes
Demonstrating DNA looping the loop, and it seems more complex than systems in which
At the time of the discovery of a site required for repression the regulatory protein binds immediately alongside RNA
and lying hundreds of base pairs upstream from its target polymerase or competes with polymerase binding. Several
promoter, no convincing mechanism of action was known. reasons can be advanced (Schleif, 1987, 1988).
Among the possibilities, however, was DNA looping in The first is that the cooperativity generated by DNA
which the AraC protein bound at the upstream repression looping allows a high occupancy of the binding sites, even
site interacted with an unknown component of the promo- though the affinity of the binding protein for each indivi-
ter or with the transcription initiation complex bound at the dual DNA site is quite low. The cooperativity arises because
promoter. Helical twist experiments (Dunn et al., 1984; Lee the binding of the protein to one of the two looping sites
& Schleif, 1989; Fig. 4), provided strong evidence in favor of holds the protein in the vicinity of the other binding site,
this idea. In these experiments, small insertions and dele- thus increasing the protein’s effective concentration in the
tions of known size were introduced between the upstream vicinity of the second site. It is the bridging that increases the
repression site and the sites required for induction. These binding. (It is an occasionally held misconception that
insertions and deletions cyclically affected the ability of the multiple binding sites for a protein increase the solution
upstream site to effect repression with a period of 10–11 bp. concentration of the protein in their vicinity. This is not so,
That is, insertions or deletions of 11, 22, and 33 bp could still as the solution concentration, that is, the concentration of
repress normally, whereas insertions or deletions of 5, 15, protein free in solution and not bound to a DNA-binding
27 bp, etc. could not. Subsequent in vivo footprinting, site, cannot be altered by the presence of nearby sites
mutational analysis, and in vitro DNA experiments provided without violating the laws of thermodynamics.)
c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved
AraC and ara regulation 785
The cooperativity inherent in DNA looping means that at The half-sites I1, O2, and I2 are involved in DNA looping
other sites, locations where looping is not possible, the and unlooping. The relative affinities of AraC for these three
binding of the protein will be very low. That is, DNA looping sites are roughly 1000, 100, and 10 (Seabold & Schleif, 1998).
allows multiple DNA-binding sites to be used to increase the As would be expected, changing the sequence of the O2 half-
sequence specificity of DNA binding. This effect may be site to that of I1 largely locks AraC in the looped state and
particularly important in eukaryotic cells, where thousands prevents induction. Analogously, changing I2 to bind AraC
of different regulatory proteins must all be present in the more tightly makes the operon constitutive and eliminates
nucleus. Hence, the concentration of each one is very low. If the requirement for arabinose for induction.
a protein is to bind at a gene’s regulatory site, the protein’s
affinity for that site must be high. Unfortunately, a protein
The activity of AraC and CAP in activating
with such a high affinity for a site will bind so tightly that its
dissociation may be so slow as to interfere with other
transcription
that some other part of RNA polymerase contacts the AR3 The extent of nature’s use of light switch-type mechan-
region. Such an interaction seems plausible because the isms involving N-terminal or C-terminal arms for con-
CAP–AraC–RNAP complex must substantially bend the trolled inter- or intra-domain interactions is not known,
DNA and it is very likely that in the complex CAP is but arm–domain interactions are widespread in nature
positioned close to RNA polymerase despite the fact that (Pabo et al., 1982; Mak & Johnson, 1993; Lee et al., 1996;
the binding site for AraC lies between the binding sites for Russo et al., 1996; Berdis et al., 1997; Luger et al., 1997; Tai
CAP and RNA polymerase. et al., 1999; ter Haar et al., 2000; de Beer et al., 2000; Vetter &
Wittinghofer, 2001).
The light switch mechanism of AraC
explains looping and unlooping Summary of the evidence for the light
Can we understand, at the atomic level, why AraC, in the
switch mechanism
c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved
AraC and ara regulation 787
c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved
AraC and ara regulation 789
2008). Such a result might be consistent with a light dictions were tested with biochemical studies that identified
switch-type mechanism operating in XylS in which the specific amino acid–nucleotide interactions using the miss-
presence of a ligand frees the DNA-binding domain of XylS ing contact method (Brunelle & Schleif, 1989). The crystal-
from the dimerization domain. To have detected an interac- lization and structure determination of MarA and Rob while
tion at the micromolar concentrations of the two domains bound to DNA (Rhee et al., 1998; Kwon et al., 2000)
that were used in the experiments, however, the domain– provided definitive answers. These are two proteins with a
domain interaction strength would have to be considerably sufficiently high homology to the DNA-binding domain of
tighter than 1 mM. Then, upon binding of the ligand, the AraC to be confident that all three proteins share a similar
interaction would have to weaken to the millimolar range (if tertiary structure. The MarA and Rob structures showed
the length of the interdomain linker in XylS is similar to that that both DNA-contacting regions in each of these proteins
found in AraC). Perhaps the simplest test of these striking were helix-turn-helix motifs.
findings would be measurements of the effects that the In the crystal structures of MarA and Rob, the two helix-
significant similarity to AraC. Because most of these possess Table 1. Homologs of AraC in the NCBI database as of fall 2009, their
detectable sequence similarity only over the coding region similarity to AraC and local gene order
for the DNA-binding domain of AraC, almost surely most of NCBI Number
these regulate genes other than those coding for the uptake similarity residues ara gene
and catabolism of arabinose. A sizeable fraction of those score to coli identical to order near
E. coli AraC AraC
w
whose function is known or can be inferred seem to be Bacterial strain AraC
involved in the virulence or the control of the expression of Escherichia coli 611 292/292 CBAD
extracellular proteins. Some of these may be regulated not by Shigella boydii 607 292 CBAD
small molecules such as arabinose, but by other proteins that Citrobacter sp. 30_2 594 275 CBAD
bind to the AraC ortholog (Plano, 2004). Salmonella typhimurium 555 270 CBAD
Quite a number of the AraC orthologs possess detectable LT2
similarity to the E. coli AraC protein over both the dimeriza- Klebsiella pneumoniae 538 266 CBAD
c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved
AraC and ara regulation 791
Fig. 8. Sequence alignments of homologs of AraC in which the fully conserved regions have a gray background.
contacting surface, S207, R178, Q230, and R277, are good bound AraC, it is not possible to tell whether the highly
candidates for contacting the dimerization domain or RNA conserved residue D257 contacts DNA or is likely to estab-
polymerase. Without knowledge of the structure of DNA- lish an important non-DNA interaction.
The regulatory protein of melibiose catabolism genes, Analogously, for the rhamnose regulators, RhaR and
MelR, possesses a clearly significant sequence similarity to RhaS, the corresponding residue is highly, but not abso-
AraC only over the DNA-binding domain. MelR orthologs lutely, conserved. Thus, residue 277 of AraC is identified
in the NCBI database selected by the same criteria as used as likely to be involved in important contacts to RNA
above for identifying orthologs of AraC also completely polymerase.
conserve the arginine corresponding to R277 of AraC. RhaR and RhaS appear to be somewhat distant relatives
of AraC. Their DNA-binding domains possess a clear
similarity to AraC, but their N-terminal regions, which
correspond to the dimerization and arabinose-binding
domain in AraC, do not possess convincing sequence
similarity to AraC. Because, however, the DNA-binding
domain of AraC alone can activate transcription from ara
c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved
AraC and ara regulation 793
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c 2010 Federation of European Microbiological Societies FEMS Microbiol Rev 34 (2010) 779–796
Published by Blackwell Publishing Ltd. All rights reserved