Biochemical and Physiological Properties of the DNA Binding Domain of AraC Protein

Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21204, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 08/2004; 340(4):731-8. DOI: 10.1016/j.jmb.2004.05.018
Source: PubMed

ABSTRACT Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.

1 Follower
18 Reads
  • Source
    • "Binding of AraC to a single I 1 half-site is much weaker than to a pair of adjacent half-sites as is found at the ara p BAD promoter. Consequently, the dissociation rate of AraC from a single half-site is much faster than from a pair of half-sites, and use of the gel-binding assay to detect binding or to measure binding constants then becomes problematic (Timmes et al., 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    FEMS microbiology reviews 09/2010; 34(5):779-96. DOI:10.1111/j.1574-6976.2010.00226.x · 13.24 Impact Factor
  • Source
    • "That the predicted expression is higher at early time points than measured suggests that there are elements of repression via AraC that the model does not reproduce. AraC has been demonstrated to be required to initiate transcription by helping to recruit RNA polymerase to the -35 site [17,18,20,41,42]; however, the protein is always present at I2 which is immediately adjacent to the -35 site. Therefore, changes in concentration of AraC, which would be measured by our reporter, may happen long after the resident AraC has fulfilled its role. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Most modelling efforts of transcriptional networks involve estimations of in vivo concentrations of components, binding affinities and reaction rates, derived from in vitro biochemical assays. These assays are difficult and in vitro measurements may not approximate actual in vivo conditions. Alternatively, changes in transcription factor activity can be estimated by using partially specified models which estimate the "hidden functions" of transcription factor concentration changes; however, non-unique solutions are a potential problem. We have applied a synthetic biology approach to develop reporters that are capable of measuring transcription factor activity in vivo in real time. These synthetic reporters are comprised of a constitutive promoter with an operator site for the specific transcription factor immediately downstream. Thus, increasing transcription factor activity is measured as repression of expression of the transcription factor reporter. Measuring repression instead of activation avoids the complications of non-linear interactions between the transcription factor and RNA polymerase which differs at each promoter. Using these reporters, we show that a simple model is capable of determining the rules of integration for multiple transcriptional inputs at the four promoters of the arabinose catabolic pathway. Furthermore, we show that despite the complex and non-linear changes in cAMP-CRP activity in vivo during diauxic shift, the synthetic transcription factor reporters are capable of measuring real-time changes in transcription factor activity, and the simple model is capable of predicting the dynamic behaviour of the catabolic promoters. Using a synthetic biology approach we show that the in vivo activity of transcription factors can be quantified without the need for measuring intracellular concentrations, binding affinities and reaction rates. Using measured transcription factor activity we show how different promoters can integrate common transcriptional inputs, resulting in distinct expression patterns. The data collected show that cAMP levels in vivo are dynamic and agree with observations showing that cAMP levels show a transient pulse during diauxic shift.
    BMC Systems Biology 06/2010; 4(1):75. DOI:10.1186/1752-0509-4-75 · 2.44 Impact Factor
  • Source
    • "The apparent Keq for the PexsC, PexsD and PexoT promoters was calculated as 1.1 Ϯ 0.2 nM, 4.1 Ϯ 0.2 nM and 5.4 Ϯ 0.6 nM respectively. This relatively high binding affinity is similar to that of other well-characterized transcriptional activators (Kwon et al., 2000; Timmes et al., 2004) and suggests that a significant fraction of the purified ExsAHis is in an active form. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is activated by ExsA, a member of the AraC/XylS family of transcriptional regulators. In the present study we examine the DNA-binding properties of ExsA. ExsA was purified as a histidine-tagged fusion protein (ExsA(His)) and found to be monomeric in solution. ExsA(His) specifically bound T3SS promoters with high affinity as determined by electrophoretic mobility shift assays (EMSA). For each promoter tested two distinct ExsA-DNA complexes were detected. Biochemical analyses indicate that the higher-mobility complex consists of a single ExsA(His) molecule bound to DNA while the lower-mobility complex results from the binding of two ExsA(His) molecules. DNase I protection assays demonstrate that the ExsA(His) binding site overlaps the -35 RNA polymerase binding site and extends upstream an additional approximately 34 bp. An alignment of all 10 ExsA-dependent promoters revealed a number of highly conserved nucleotides within the footprinted region. We find that most of the highly conserved nucleotides are required for transcription in vivo; EMSA-binding assays confirm that several of these nucleotides are essential determinants of ExsA(His) binding. The combined data support a model in which two ExsA(His) molecules bind adjacent sites on the promoter to activate T3SS gene transcription.
    Molecular Microbiology 06/2008; 68(3):657-71. DOI:10.1111/j.1365-2958.2008.06179.x · 4.42 Impact Factor
Show more


18 Reads
Available from