Repression by AraC

Repression by AraC

In the presence of arabinose, AraC protein induces expression of the metabolic and active transport genes. That is, it is a positive regulator. Most unexpectedly, AraC protein also appears to repress expression of these genes. Three types of experiments demonstrate the repression exerted by AraC protein. The simplest utilizes Ara⁺ revertants that are isolated in strains deleted of the araC gene. These mutations are called I^c. They lie in the p_BAD RNA polymerase-binding site and they permit a low rate of polymerase binding and initiation in the absence of AraC protein. Repression is revealed by the fact that the constitutive promoter activity of the I^c mutants is reduced by the presence of AraC protein. The protein can act in trans to repress just like the lac repressor.

Experiments with the I^c mutations also show that at least part of the site required for repression of p_BAD lies upstream from all of the sites required for induction. This is shown by the properties of strains containing the two deletions, ∆1 or ∆2. ∆1 extends just to the end of the araC gene and∆2 ends beyond the araC gene, so that half of theregulatory region between the araC and araBAD genes containing p_BAD has been deleted. The promoter p_BAD in both of the strains is undamaged

by the deletions because it remains fully inducible when AraC protein is provided in trans. Repression of the I^c mutation by AraC protein occurs only in the strain containing D1 (Table 12.2). Therefore at least part of the site required for repression has been deleted by D2.

Conceivably, the repression exerted by AraC occurs only in the I^c mutants. Therefore experiments will be described that do not use the I^c mutations. The experiments use the same two deletions as described above, D1 and D2. AraC⁺D(BAD) episomes are introduced into the deletion strains. When these cells are grown in the absence of arabinose, the basal level in the strain containing D1 is normal but the basal level in the strain containing D2 is 10 to 30 times normal. This result confirms the repression effect and also shows that in the absence of arabinose, a little of the AraC protein is in the inducing state and can weakly induce p_BADif the region defined byD2 has been deleted.

A third demonstration of repression in the ara system utilizes araC constitutive mutations, araC^c. This type of mutation causes the arabi-nose enzymes to be induced even in the absence of the normal inducer, L-arabinose. Diploids containing both araC^c and araC⁺ mutations are surprising, for the C⁺ allele is almost completely dominant to the C^c allele (Table 12.3). C⁺/C^c diploids possess nearly the normal uninduced level of arabinose enzymes in the absence of arabinose and nearly the fully induced level of enzymes in the presence of arabinose. In light of the other experiments showing repression, these results are most simply explained as resulting from repression by the C⁺ protein despite the presence of the C^c protein. These results, however, are also consistent with AraC protein being an oligomer in which the in vivo dominance of C⁺ results from subunit mixing.

Figure 12.7 The presence of arabinose drives AraC protein into the inducingstate, which acts positively at the I site. Before arabinose addition, the protein was acting negatively through the O site.

The araC^c mutants mentioned above are easily isolated with the aid of the arabinose analog 5-methyl-L-arabinose, which is also known as D-fucose. D-fucose cannot be metabolized by E. coli, but it does interact with AraC protein to inhibit the normal induction by arabinose. Mutants able to grow on arabinose in the presence of fucose are called araC^c

The results described above indicate that AraC protein can induce or repress the initiation of transcription of the arabinose operons in E. coli. AraC protein must therefore exist in at least two states, a repressing and an inducing state, and arabinose must drive the population of AraC protein molecules in a cell toward the inducing state (Fig. 12.7). The deletions show that the most upstream site required for repression lies further upstream than the most upstream site required for induction. By analogy to the lac operon, the site required for repression is called an operator, O. The I site stands for induction.