Activation of ara operons by a truncated AraC protein does not require inducer.

Abstract

The araC gene of Escherichia coli encodes a protein that binds the inducer L-arabinose to activate the transcription of three ara operons. In a study to determine the functional domains within the AraC protein, we have generated a set of overlapping deletions from the proximal end of the araC gene. We found that the removal of up to nearly 60% of the coding sequence of this protein still allows transcriptional activation of the ara operons in vivo, up to 27% that of the wild type. These truncated proteins, however, no longer require arabinose for induction. The ligand-induced conformational change apparently either releases or unmasks an existing functional domain within AraC, rather than generating a new conformation that is required for activation of the promoter of araBAD. Since the truncated protein ofthe mutant C154 (which lacks 153 amino acid residues from the N terminus) retains DNA binding specificity, the DNA-recognition domain is localized in the C-terminal half of the AraC protein. Truncated proteins were unable to repress araBAD or araC in vivo, even though they were able to bind all ara operators. We propose that the N-terminal half of AraC is essential for the formation of the DNA loops that are responsible for repression of araBAD and for autoregulation of araC.Proteins that activate multiple transcription units are widely distributed among biological systems-procaryotic (1), eucaryotic (2), and viral (3). This arrangement may reflect an advantage to the cellular economy in being able to control the synthesis of many genes by a single regulatory protein (1). In addition, when the transcriptional activator undergoes a change in function by binding a small molecule, the cell has an economical way of controlling the expression of a large number of genes in response to an effector molecule in the environment.The araC gene of Escherichia coli encodes a protein that binds the inducer L-arabinose to activate the transcription of three widely separated ara operons. Two of these encode proteins involved in arabinose transport, and the third, the araBAD operon, encodes enzymes of arabinose catabolism (4-6). All three ara operons are preceded by common DNA sequences that are tandem repeats of a 17-base-pair ( (Fig. 1) (7). In this manner, the regulatory protein AraC signals the presence of the metabolizable substrate in the medium and initiates the synthesis of proteins required for its utilization.The AraC protein is biologically active in both ligandbound and ligand-free forms. In the absence of the inducer, AraC is capable of activating PBAD to about 1% that of the induced level. This 1% activation by AraC without inducer is repressed by the binding of AraC to a far upstream lowaffinity operator (araO2) (4,8). Furthermore, it has been shown that repression of PBAD in the wild type requires not only araO2 but also the high-affinity araI site; it was proposed that repression requires an association between AraC molecules bound at araO2 and araI that loops out 20 turns of intervening DNA (9). This i...

PchR, a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor

“…Although the exact mechanism by which arabinose causes induction of the system is unclear, it appears that arabinose interacts directly with AraC, causing a conformational change in the protein which ameliorates loop formation and enhances binding to adjacent half sites (araI 1 and araI 2 ) whose occupancy facilitates ara gene expression (35,36). Evidence suggests that arabinose (the so-called effector) interacts with the N-terminal portion of the protein, while the C-terminal domain mediates binding to the DNA (37). This property appears to be shared by other members of the AraC family of regulators (45).…”

Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor.

“…The activity of several regulatory proteins is masked by an inhibitory domain on the same protein (Godowski et al 1987;Hollenberg et al 1987;Menon and Lee 1990;Choi and Greenberg 1991). For these proteins, deletion of the inhibitory region results in a constitutively active transcription factor.…”

“…The steroid hormone binding domain of the glucocorticoid receptor inhibits this protein's activity as a transcriptional enhancer in the absence of hormone (Godowski et al 1987;Hollenberg et al 1987). The AraC and LuxR transcriptional activators contain amino-terminal regions that inhibit the ability of these proteins to activate transcription in the absence of their respective inducer molecules (Menon and Lee 1990;Choi and Greenberg 1991). In addition, the major cr factor of Escherichia coli contains a region that masks the ability of free cr z~ to bind DNA (Dombroski et al 1992), and inhibition of DNA binding by this region is presumably relieved by interaction of cr with core RNA polymerase.…”

Aromatic ligand binding and intramolecular signalling of the phenol‐responsive σ54‐dependent regulator DmpR

“…Repression is a common mechanism by which the intrinsic activity of different families of prokaryotic transcriptional activators are kept silent until the activities of the genes they control are required. For example, the normal activating signal is not required when various portions of AraC (Menon and Lee, 1990), FixJ (Kahn and Ditta, 1991), LuxR (Choi and Greenberg, 1991) or Spo0A (Ireton et al, 1993) are deleted. Similarly, the DNA binding properties of 70 and related sigma factors are also under repression control (Dombroski et al, 1993).…”