Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r

Abstract

Mutations in the araC gene have been isolated which alter both the activator and autoregulatory functions of AraC protein (L. G. Cass and G. Wilcox, J. Bacteriol. 166:892-900, 1986). In this study, the effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the presence of L-arabinose. The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which was not observed in the araC+ cell. Experiments were therefore focused on understanding the mechanism of this novel activation. We describe a systematic analysis of the effect of mutations within the known regulatory binding sites for araBAD and araC transcription on araC expression. Our results suggest that the novel activation of araC expression requires the AraC activator-binding site, aral, and the cyclic AMP receptor protein-cyclic AMP complex-binding site. We also found that in the absence of L-arabinosej the aral site was required for maximal autoregulation by the wild-type AraC protein.The AraC protein functions as both a positive and negative regulator of gene expression in Escherichia coli (8,17). Transcription of the araBAD operon is repressed by AraC protein in the absence of L-arabinose and is activated by AraC protein in the presence of L-arabinose (8, 17). In the presence or absence of L-arabinose, the araC gene is autogenously regulated (2,12,19). Maximal expression of both the araBAD operon and the araC gene requires the presence of the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) (8, 17).The araBAD operon and araC gene promoters are separated by 147 base pairs (bp) (33) and are transcribed in opposite directions (34). The DNA sequence of this ara regulatory region is known (10, 30). Genetic and biochemical analyses have defined the binding sites for the regulatory proteins AraC and CRP and for RNA polymerase (6, 19, 28) (Fig. 1). Based on these studies, models for the regulation of the araCBAD gene complex have been proposed, aspects of which are summarized as follows. (Fig. 1).The Escherichia coli araC gene encodes a 292-amino-acid polypeptide, as predicted by DNA sequence analysis (23). Mutations in the araC gene have been isolated which affect both the activator and autoregulatory functions of AraC * Corresponding author. t Present address: Department of Biological Sciences, University of California, Santa Barbara, CA 93106. protein (5). The effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the absence of L-arabinose. Previous studies have shown that in the wildtype cell, induction of araC expression by L-arabinose does not occur (2). In the present study, we have reexamined the effect of these araC mutations on autoregulation in the presence of L-arabinose. The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which is not observed in the araC+ cell. Therefore, experiments were focused on understanding the mechanism of this novel activation. We describe a systematic...

Leafy Gall Formation Is Controlled by fasR , an AraC-Type Regulatory Gene in Rhodococcus fascians

“…Because AraC-type transcriptional regulators are often autoregulatory (8,25), a transcriptional fusion of the upstream region of fasR to uidA was constructed (pRFDV6) (Fig. 1D).…”