Active role of the interdomain linker of AraC

Abstract

Mutations in the interdomain linker of the gene for the AraC regulatory protein of Escherichia coli that severely interfere with the protein's ability either to repress or to activate transcription have been found. These mutations have relatively small effects on the dimerization domain's ability to bind arabinose or to dimerize the protein or on the DNA-binding domain's affinity for a single DNA half-site. The linker mutations, however, dramatically change the affinity of AraC for binding to two direct-repeat DNA half-sites. Less dramatically, the induction-deficient linker variants also display altered DNA sequence selectivity. These results show that changing the sequence of the interdomain linker can profoundly affect the dimerization domain-DNA-binding domain interactions in AraC. The smaller effects on the functions of the individual domains could be the direct result of the linker alterations but more likely are the indirect result of the altered dimerization domain-DNA-binding domain interactions. In summary, the linker does not simply function as a passive and flexible connector between the domains of AraC but, instead, is more directly involved in the protein's dimerization domain-DNA-binding domain interactions.

Fig. 1.

The repressing and inducing states of the araCBAD regulatory region and the domain organization of a single subunit of the AraC homodimer showing the locations of the important structural features. CAP, catabolite gene activator protein; RNAP, RNA polymerase.

Fig. 2.

Sequence of the interdomain linker region of AraC, the mutations isolated, and their resulting phenotypes. Gray shading, wild-type induction and repression behavior; light gray shading, Rep⁻ or Ind⁻.

Fig. 3.

Binding of arabinose by wild-type AraC and Rep⁻ mutant proteins. The average emission wavelength of the intrinsic tryptophan fluorescence of AraC or a Rep⁻ variant as a function of molar arabinose concentration.

Fig. 4.

Binding of an I₁ half-site by wild-type AraC and the Rep⁻ variants. The fluorescence anisotropy of a Cy5-labeled I₁ half-site as a function of the total molar concentration of WT AraC or a Rep⁻ variant.

Fig. 5.

A typical electrophoretic mobility shift assay experiment to measure the affinity of AraC for direct-repeat I₁-I₁ half-sites. Cy5-labeled I₁-I₁ DNA was incubated with increasing amounts of WT AraC. The different migration rates are due to time differences between when the first 8 lanes and the last 7 lanes were loaded, which resulted in different lengths of time for the electrophoresis. The fraction bound was fit to equation 1, and for the experiment shown, the K_d of WT AraC binding to I₁-I₁ DNA in 500 mM KCl binding buffer was 2.7 nM.

Fig. 6.

Binding of I₁-I₁ direct-repeat DNA by wild-type AraC and its linker variants. Binding of WT AraC (A and B), Rep⁻ AraC (A), and Ind⁻ AraC (B) to Cy5-labled I₁-I₁ direct-repeat DNA in the absence of arabinose (A) and in its presence (B) as a function of the molar concentration of KCl. Each data point represents the K_d (M) from fitting the fraction bound and the error associated with the fit of a single titration curve to equation 1. The lines represent the best fits of the data points to straight lines, as described previously (22).