The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araCprotein-arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it simultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.Studies on the L-arabinose operon of Escherichia coli have established the following important facts on regulation of the divergently oriented ara promoters PC and PBAD (see Fig. 1).The activity of both promoters is stimulated by the cyclic AMP (cAMP) receptor protein (CRP) in the presence of cyclic AMP (1-4). The promoter PBAD is positively regulated by araC protein in the presence of arabinose-i.e., the protein is required for activity of the promoter (1, 2, 5). Under noninducing conditions, the araC protein instead acts negatively to repress both the PC and PBAD promoters (1-3, 6). At least part of the DNA site necessary for repression of PBAD lies upstream from all of the sites necessary for induction of PBAD, as shown by the existence of deletions entering the ara regulatory region from the Pc side that abolish repression of PBAD without affecting induction of PBAD (6, 7).The requisite components are now available for in vitro studies of the mechanism of regulation of the ara operon. The regulatory region DNA has been isolated, and its nucleotide sequence has been determined (8,9) and found to contain elements similar to the RNA polymerase-binding sites seen in other E. coli promoters at about 10 and 35 bases before the start sites of transcription (8). The sequence also contains several stretches that resemble the CRP-binding site in the gal operon (10). Also available are the proteins involved in the regulation of the ara operon: araC protein (11), CRP (12), and RNA polymerase (13).In the work reported here, we have utilized the DNA se- MATERIALS AND METHODS DNA fragments for sequence determination and protection were obtained from plasmid pMB9ara440 (8) by EcoRI endonuclease digestion and polyethylene glycol precipitation (16). CRP was purified as described (12), and ara...

We isolated a colicin K-insensitive energy uncoupled mutant of Escherichia coli . This mutant was presumed to be an ecf mutant as evidenced by its similarity to a known ecf mutant (M. A. Lieberman and J.-S. Hong, 1974) with respect to the mutational site, reversion pattern, and defects in transport and growth. The mutation conferring the colicin K-insensitivity resided in the ecf gene as the majority of the secondary mutations overcoming the ecf phenotype reverted the colicin K-insensitive phenotype to colicin K-sensitive. The insensitivity of the mutant to colicin K was not due to either a defect in adsorption or to a lack of the energized membrane state. The defect was most probably due to the inability of colicin K molecules to interact with their target. Our previous studies concerning the role of the ecf gene product in energy coupling to active transport and oxidative phosphorylation support the contention that the ECF protein is itself the direct target of colicin K.