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PubMed · 12596232

AraC protein: a love-hate relationship.

Robert Schleif
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Abstract

In the bacterium Escherichia coli, the AraC protein positively and negatively regulates expression of the proteins required for the uptake and catabolism of the sugar L-arabinose. This essay describes how work from my laboratory on this system spanning more than thirty years has aided our understanding of positive regulation, revealed DNA looping (a mechanism that explains many action-at-a-distance phenomena) and, more recently, has uncovered the mechanism by which arabinose shifts AraC from a state where it prefers to bind to two well-separated DNA half-sites and form a DNA loop to a state where it binds to two adjacent half-sites and activates transcription. This work required learning how to assay, purify, and work with a protein possessing highly uncooperative biochemical properties. Present work is focussed on understanding arabinose-responsive mechanism in atomic detail and is also directed towards understanding protein structure and function well enough to be able to engineer the allosteric mechanism seen in AraC onto other proteins.

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AraC Transcription FactorArabinoseBacterial ProteinsDNAEscherichia coliEscherichia coli ProteinsGene DeletionLightModels, BiologicalModels, GeneticProtein BindingProtein Structure, TertiaryRepressor ProteinsTime FactorsTranscription FactorsJournal ArticleResearch Support, U.S. Gov't, P.H.S.Review

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Robert Schleif. 2003. AraC protein: a love-hate relationship.. https://doi.org/10.1002/bies.10237

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PubMed2007

Structure and properties of a truely apo form of AraC dimerization domain.

The arabinose-binding pockets of wild type AraC dimerization domains crystallized in the absence of arabinose are occupied with the side chains of Y31 from neighboring domains. This interaction leads to aggregation at high solution concentrations and prevents determination of the structure of truely apo AraC. In this work we found that the aggregation does not significantly occur at physiological concentrations of AraC. We also found that the Y31V mutation eliminates the self-association, but does not affect regulation properties of the protein. At the same time, the mutation allows crystallization of the dimerization domain of the protein with only solvent in the arabinose-binding pocket. Using a distance difference method suitable for detecting and displaying even minor structural variation among large groups of similar structures, we find that there is no significant structural change in the core of monomers of the AraC dimerization domain resulting from arabinose, fucose, or tyrosine occupancy of the ligand-binding pocket. A slight change is observed in the relative orientation of monomers in the dimeric form of the domain upon the binding of arabinose but its significance cannot yet be assessed.

AraC Transcription Factor↗
PubMed2006

Transcriptional regulation of the pdt gene cluster of Pseudomonas stutzeri KC involves an AraC/XylS family transcriptional activator (PdtC) and the cognate siderophore pyridine-2,6-bis(thiocarboxylic acid).

In order to gain an understanding of the molecular mechanisms dictating production of the siderophore and dechlorination agent pyridine-2,6-bis(thiocarboxylic acid) (PDTC), we have begun characterization of a gene found in the pdt gene cluster of Pseudomonas stutzeri KC predicted to have a regulatory role. That gene product is an AraC family transcriptional activator, PdtC. Quantitative reverse transcription-PCR and expression of transcriptional reporter fusions were used to assess a role for pdtC in the transcription of pdt genes. PdtC and an upstream, promoter-proximal DNA segment were required for wild-type levels of expression from the promoter of a predicted biosynthesis operon (P(pdtF)). At least two other transcriptional units within the pdt cluster were also dependent upon pdtC for expression at wild-type levels. The use of a heterologous, Pseudomonas putida host demonstrated that pdtC and an exogenously added siderophore were necessary and sufficient for expression from the pdtF promoter, i.e., none of the PDTC utilization genes within the pdt cluster were required for transcriptional signaling. Tests using the promoter of the pdtC gene in transcriptional reporter fusions indicated siderophore-dependent negative autoregulation similar to that seen with other AraC-type regulators of siderophore biosynthesis and utilization genes. The data increase the repertoire of siderophore systems known to be regulated by this type of transcriptional activator and have implications for PDTC signaling.

AraC Transcription Factor↗
PubMed2006

Specific interactions by the N-terminal arm inhibit self-association of the AraC dimerization domain.

Deletion of the regulatory N-terminal arms of the AraC protein from its dimerization domain fragments increases the susceptibility of the dimerization domain to form a series of higher order polymers by indefinite self-association. We investigated how the normal presence of the arm inhibits this self-association. One possibility is that arms can act as an entropic bristles to interfere with the approach of other macromolecules, thereby decreasing collision frequencies. We examined the repulsive effect of flexible arms by measuring the rate of trypsin cleavage of a specially constructed ubiquitin-arm protein. Adding an arm to ubiquitin or increasing its length produced only a modest repulsive effect. This suggests that arms such as the N-terminal arm of AraC do not reduce self-association by entropic exclusion. We consequently tested the hypothesis that the arm on AraC reduces self-association by binding to the core of the dimerization domain even in the absence of arabinose. The behaviors of dimerization domain mutants containing deletions or alterations in the N-terminal arms substantiate this hypothesis. Apparently, interactions between the N-terminal arm and the dimerization domain core position the arm to interfere with the protein-protein contacts necessary for self-association.

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