The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

Abstract

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...

Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase

“…The lac operon (which apparently has only one mRNA start site) requires but one CAP molecule, bound at -50 to -70, for transcriptional stimulation (4,16). CAP binds between positions -80 and -100 at the araBAD operon; however, the araC protein, which binds between CAP and RNA polymerase, is also necessary for expression of the operon (26). There are strildng sequence homologiesbetween the CAPbinding sites of gal (-30'to -44 and -48 to -75) (2,22), lac (-50 to -75) (7), and araBAD (-82 to -92) (27,28).…”

Spacing mutations between theEscherichia coliPBAD RNA polymerase binding site and thearaC(I) induction site

“…Both the level of protein required for binding and the pattern of protection from nuclease digestion remain unchanged, however it is not yet known whether these experiments, under the conditions employed, could detect some physiologically important alterations in binding. While all the mutations lie just within the DNA region protected by araC protein (7,8), the insertion mutations duplicate DNA sequence so that the binding sites remain largely intact (Fig. 2) (20,21).…”

“…DNase I protection and galactokinase assay DNase I protection was as described (7). Approximately 20 ng of ara DNA fragment was incubated for 10 minutes at 370 with araC protein in 100 4l 20 mM Tris-HCl pH 7.5, 50 mM KC1, 2.5 mM MgC12, 0.1 mM DTT, 100 mM L-arabinose and 25 pg/ml BSA.…”

“…Both pC and PBAD are stimulated by the cAMP receptor protein (CRP)-cAMP complex (2)(3)(4)6). Between the divergently oriented promoters, the DNA binding sites for these effectors occupy a 147 base pair noncoding region in the order: RNA polymerase at PCs which overlaps araC protein repressor (0); cAMP receptor protein; araC protein inducer (I); and RNA polymerase at PBAD (7,8).…”

Conformational selection of syn‐cAMP upon binding to the cAMP receptor protein

“…The cAMP. CRP complex interacts with specific DNA sites at or near promotors [6,7], stimulating the initiation of mRNA synthesis [8,9]. The mechanism of the activation of transcription, however, is still poorly understood.…”