Binding Sites of the ara Regulatory Proteins

Binding Sites of the ara Regulatory Proteins

Now that the regulatory phenomena of the ara operon have been laid out, what can be said about its mechanism of regulation? The nucleotide sequences of the promoters p_BAD, p_E, p_FGH, show three distinct regions of homology among themselves. One of these is the RNA polymerase-binding site. Another is the cyclic AMP receptor protein-binding site, and the third is the AraC protein-binding site. Inexplicably, the araBAD

Figure 12.10 Schematic of the binding sites of AraC protein, CRP, and RNApolymerase on the araBAD regulatory region. The araO1 site is the operator for pCand the araI site is required for induction.

RNA polymerase-binding site is quite similar to the RNA polymerase sites of promoters that do not require auxiliary proteins for activity. Since p_BAD requires the auxiliary proteins AraC and CRP for its activity, the polymerase-binding sequence might have been expected to be mark-edly different from the consensus RNA polymerase-binding sequence.

The identity of the protein-binding sites was established by DNAse footprinting. In araBAD, the AraC protein-binding site is just upstream from RNA polymerase, and the CRP-binding site is just upstream from that of araC (Fig. 12.10). The AraC-binding site is called araI for induction. These three sites are required for induction of p_BAD.

A second AraC protein-binding site called araO₁ lies another 60 nucleotides upstream of araI. This site overlaps the RNA polymerase-binding site of p_C, and since occupancy of araO₁ by AraC protein sterically interferes with the binding of RNA polymerase at p_C, araO₁ is an operator of p_C. AraO₁ is not directly involved with repression of p_BAD. A simple demonstration of this is the behavior of p_C and p_BAD as the level of AraC protein is increased in a series of strains containing plasmids with araC fused to promoters of different strengths. As the level of AraC protein is increased, pC shuts down but pBAD remains fully inducible (Table 12.4).

Deletion analysis showed that one of the sites required for repression of p_BAD lies beyond araO₁. It is called araO₂, and it lies 270 base pairs upstream from the p_BAD transcription start point. Footprinting has confirmed that AraC protein binds to this site, and mutation analysis

has shown that a single nucleotide change in this region can eliminate repression of p_BAD. Deletion of this site is the reason that the original deletion that was isolated by Englesberg could no longer repress p_BAD but could induce. His deletion extended through araO₂ into araO₁, and during the interval of several years between the discovery of O₁ and the discovery of O₂, araO₁ was thought to be required for repression of p_BAD.

The location of the CRP site required for stimulation of the p_BAD promoter is most surprising. In the lac operon, the CRP site lies at positions -48 to -78 with respect to the start of transcription, just next to RNA polymerase. At the ara promoter, AraC protein occupies this site and CRP lies further away, binding to positions -80 to -110. CRP makes specific contacts with RNA polymerase in activating transcription of the lac promoter. Does it make a different set of specific contacts with AraCprotein, and does AraC protein make yet another set of contacts with RNA polymerase? Alternatively, both proteins might simultaneously contact RNA polymerase to activate transcription, but it seems more likely that CRP activates through a DNA bending mechanism and that AraC activates through direct protein-protein contacts.