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The crystal structure of the arabinose-binding and dimerization domain of the Escherchia coli gene regulatory protein AraC was determined in the presence and absence of L-arabinose. The 1.5 angstrom structure of the arabinose-bound molecule shows that the protein adopts an unusual fold, binding sugar within a beta barrel and completely burying the arabinose with the amino-terminal arm of the protein. Dimer contacts in the presence of arabinose are mediated by an antiparallel coiled-coil. In the 2.8 angstrom structure of the uncomplexed protein, the amino-terminal arm is disordered, uncovering the sugar-binding pocket and allowing it to serve as an oligomerization interface. The ligand-gated oligomerization as seen in AraC provides the basis of a plausible mechanism for modulating the protein's DNA-looping properties.

DNA looping is widely used in nature. It is well documented in the regulation of prokaryotic and eukaryotic gene expression, DNA replication, and site-specific DNA recombination. Undoubtedly looping also functions in other protein-DNA transactions such as repair and chromosome segregation. While the underlying physical chemistry of DNA looping is common to all systems, the precise biochemical details of looping and the utilization of looping by different systems varies widely. Looping appears to have been chosen by nature in such a wide variety of contexts because it solves problems both of binding and of geometry. The cooperativity inherent in binding a protein to multiple sites on DNA facilitates high occupancy of DNA sites by low concentrations of proteins. DNA looping permits a sizeable number of DNA-binding proteins to interact with one of their number, for example RNA polymerase. Finally, DNA looping may simplify evolution by not requiring a precise spacing between a protein's binding site and a second site on the DNA.

A site has been found that is required for repression of the Escherichia coli araBAD operon. This site was detected by the in vivo properties of deletion mutants. In vitro protection studies with DNase I and dimethylsulfate showed that araC protein can specifically bind in this area to nucleotides lying at position -265 to -294 with respect to the ara-BAD operon promoter (PBAD) transcription start point. The previously known sites of protein binding in the ara operon lie between +20 and -160. Since the properties of deletion strains show that all the sites required for araBAD induction lie between +20 and -110, the new site at -280 exerts its repressive action over an unusually large distance along the DNA. 0,5,11,15,24, and 31 base pairs of DNA between the new site and PBAD were constructed. Repression was impaired in those cases in which half-integral turns of the DNA helix were introduced, but repression was nearly normal for the insertions of 0, +11, and +31 base pairs.The L-arabinose operon in Escherichia coli is well documented to be positively regulated by the araC protein. Additionally, the operon is negatively regulated by the same protein (1-4). Paradoxically, the negative regulation appears to involve a site lying upstream of all the sites required for induction. Initially, the site involved in this repression phenomenon appeared to be the araC-binding site, which lies from position -110 to -140 (2, 5-7) (Fig. 1). From this position, the protein could be imagined to make direct contact with the complex of cyclic AMP receptor protein araC protein-RNA polymerase, all of which are involved in induction. Since upstream repression, even from this nearby site, appeared unusual, we examined the question more carefully by using a set of deletions.As reported here, the deletions and in vitro binding experiments revealed the existence of yet another site for araC protein binding in the ara regulatory region. This site at position -280 lies too far for any simple direct interaction to exist between it and the complex of proteins on DNA that are required for initiation of transcription, and yet this site is required for repression of transcription. This finding of a second regulatory site located a considerable distance from the promoter is similar to the recent finding in the gal operon of a second operator site located downstream from the promoter and lying within the galE gene (8). MATERIALS AND METHODSMedia and Strains and General Methods. Media, strains, and general methods were as described (9-12).Construction of pTD3 and pTD4. A 440-base-pair fragment containing the araCBAD regulatory region (13) blunt-ended by treatment with S1 nuclease. HindII1 and EcoRI linkers were ligated in 10 M excess to the blunt-ended fragment, followed by codigestion with HindIII and EcoRI. The ara fragments were then ligated to HindIII/EcoRI-cut pBR322 that had been purified from the 30-base-pair fragment (14) by A-15 agarose chromatography. Plasmids having the inserted ara fragment in the desired orientation were sele...

This review covers the physiological aspects of regulation of the arabinose operon in Escherichia coli and the physical and regulatory properties of the operon's controlling gene, araC. It also describes the light switch mechanism as an explanation for many of the protein's properties. Although many thousands of homologs of AraC exist and regulate many diverse operons in response to many different inducers or physiological states, homologs that regulate arabinose-catabolizing genes in response to arabinose were identified. The sequence similarities among them are discussed in light of the known structure of the dimerization and DNA-binding domains of AraC.

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Expression of the L-arabinose BAD operon in Escherichia coli is regulated by AraC protein which acts both positively in the presence of arabinose to induce transcription and negatively in the absence of arabinose to repress transcription. The repression of the araBAD promoter is mediated by DNA looping between AraC protein bound at two sites near the promoter separated by 210 base pairs, araI and araO2. In vivo and in vitro experiments presented here show that an AraC dimer, with binding to half of araI and to araO2, maintains the repressed state of the operon. The addition of arabinose, which induces the operon, breaks the loop, and shifts the interactions from the distal araO2 site to the previously unoccupied half of the araI site. The conversion between the two states does not require additional binding of AraC protein and appears to be driven largely by properties of the protein rather than being specified by the slightly different DNA sequences of the binding sites. Slight reorientation of the subunits of AraC could specify looping or unlooping by the protein. Such a mechanism could account for regulation of DNA looping in other systems.