Three binding sites for AraC protein are required for autoregulation of araC in Escherichia coli.

Three binding sites for AraC protein were shown to be required for the autoregulation of araC: araI1, araO1, and araO2. Selective inactivation of AraC-binding sites on the DNA demonstrated that araO1 and araO2 are required in vivo to produce repression of araC in the presence of arabinose, whereas araI1 and araO2 are required in its absence. We found that the low-affinity site araO2 is essential for araC autoregulation; araO1 and araI1 provide high-affinity AraC-binding sites, which allow cooperative binding at araO2. Profound effects on the araBAD promoter and the araC promoter are produced by ligand-induced changes in AraC occupancy of functional sites on the DNA. We suggest that AraC exerts its multiplicity of controls through two alternative states of cooperative interactions with DNA and we illustrate this with a model. This model presents our interpretations of activation and repression of the araBAD operon and the autoregulation of the araC gene.     

Arabinose-induced binding of AraC protein to araI2 activates the araBAD operon promoter.

The state of Escherichia coli araI DNA occupancy by AraC protein has been found to change from a two-turn to a four-turn occupancy upon the addition of the inducer arabinose. The araI site is separable into two contiguous regions, araI1 and araI2. araI1 binds both ligand-bound and ligand-free AraC protein, whereas araI2 binds AraC protein in the presence of arabinose only. A mutation in araI and a known mutation in araC led to the loss of araI2 binding, while binding to araI1 was unaffected. Both mutants failed to activate the promoter of the araBAD operon. We propose that araI2 occupancy by AraC protein leads to RNA polymerase recognition of the araBAD promoter and that araI1 acts as a switch mechanism allowing both the repressor and the activator forms of AraC protein to regulate the araBAD promoter.     

Catabolite-Insensitive Revertants of Lac Promoter Mutants

The maximum rate of expression of the lac operon is severely reduced in lac promoter mutants. Revertants of these mutations which produce higher levels of enzyme were isolated. Some of these revertants had lost sensitivity to catabolite repression and transient repression. The mutations responsible for these losses took place at sites very close to the original promoter mutations. From these results we conclude that the promoter itself is the target site for both catabolite and transient repression of the lac operon.     

Purification and DNA-Binding Properties of the Catabolite Gene Activator Protein

A protein required for the activation of the lac operon has been extensively purified and partly characterized. This protein, called CGA protein (catabolite gene activator protein, sometimes named CAP), is a dimer with subunits of 22,000 daltons. Purified CGA protein has a substantial affinity for DNA; this affinity is greatly strengthened by cAMP and strongly inhibited by cGMP. Other studies have shown that these cyclic nucleotides compete for a binding site on CGA protein. The opposing effects of the two cyclic compounds in DNA-CGA protein binding show a parallel behavior to their effects on the expression of the lac operon. Thus cAMP, in addition to CGA protein, is required for expression of the lac operon, whereas cGMP inhibits the expression. The obvious inference is that CGA protein activates the lac operon by binding to the DNA under the influence of cAMP. Thus, CGA protein seems to be a new type of regulatory protein: a DNA-binding activator.     

The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites.

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the araI site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when araI and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are araI, araO2, and perhaps the araBAD operon RNA polymerase binding site.