A site has been found that is required for repression of the Escherichia coli araBAD operon. This site was detected by the in vivo properties of deletion mutants. In vitro protection studies with DNase I and dimethylsulfate showed that araC protein can specifically bind in this area to nucleotides lying at position -265 to -294 with respect to the ara-BAD operon promoter (PBAD) transcription start point. The previously known sites of protein binding in the ara operon lie between +20 and -160. Since the properties of deletion strains show that all the sites required for araBAD induction lie between +20 and -110, the new site at -280 exerts its repressive action over an unusually large distance along the DNA. 0,5,11,15,24, and 31 base pairs of DNA between the new site and PBAD were constructed. Repression was impaired in those cases in which half-integral turns of the DNA helix were introduced, but repression was nearly normal for the insertions of 0, +11, and +31 base pairs.The L-arabinose operon in Escherichia coli is well documented to be positively regulated by the araC protein. Additionally, the operon is negatively regulated by the same protein (1-4). Paradoxically, the negative regulation appears to involve a site lying upstream of all the sites required for induction. Initially, the site involved in this repression phenomenon appeared to be the araC-binding site, which lies from position -110 to -140 (2, 5-7) (Fig. 1). From this position, the protein could be imagined to make direct contact with the complex of cyclic AMP receptor protein araC protein-RNA polymerase, all of which are involved in induction. Since upstream repression, even from this nearby site, appeared unusual, we examined the question more carefully by using a set of deletions.As reported here, the deletions and in vitro binding experiments revealed the existence of yet another site for araC protein binding in the ara regulatory region. This site at position -280 lies too far for any simple direct interaction to exist between it and the complex of proteins on DNA that are required for initiation of transcription, and yet this site is required for repression of transcription. This finding of a second regulatory site located a considerable distance from the promoter is similar to the recent finding in the gal operon of a second operator site located downstream from the promoter and lying within the galE gene (8). MATERIALS AND METHODSMedia and Strains and General Methods. Media, strains, and general methods were as described (9-12).Construction of pTD3 and pTD4. A 440-base-pair fragment containing the araCBAD regulatory region (13) blunt-ended by treatment with S1 nuclease. HindII1 and EcoRI linkers were ligated in 10 M excess to the blunt-ended fragment, followed by codigestion with HindIII and EcoRI. The ara fragments were then ligated to HindIII/EcoRI-cut pBR322 that had been purified from the 30-base-pair fragment (14) by A-15 agarose chromatography. Plasmids having the inserted ara fragment in the desired orientation were sele...

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...

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Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species utilizing an activation-decarboxylation reaction which yields formate and CO 2 . frc, the gene encoding formyl coenzyme A transferase, an enzyme which transfers a coenzyme A moiety to activate oxalic acid, was cloned from the bacterium Oxalobacter formigenes. DNA sequencing revealed a single open reading frame of 1,284 bp capable of encoding a 428-amino-acid protein. A presumed promoter region and a -independent termination sequence suggest that this gene is part of a monocistronic operon. A PCR fragment containing the open reading frame, when overexpressed in Escherichia coli, produced a product exhibiting enzymatic activity similar to the purified native enzyme. With this, the two genes necessary for bacterial catabolism of oxalate, frc and oxc, have now been cloned, sequenced, and expressed.

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Mouse lambda-genes are rearranged in specific V lambda-JC lambda associations: V lambda 1 has only been found to rearrange with JC lambda 3 or JC lambda 1, whereas V lambda 2 mainly rearranges with JC lambda 2 and very rarely with JC lambda 1,3. In order to determine the physical basis for these associations we have cloned the respective lambda-genes and large portions of their flanking regions by chromosomal walking in a total of 141 kb of phage and cosmid clones. With the use of unique probes obtained from such clones, the order and orientation of the mouse lambda-gene segments were determined by analyzing patterns of DNA deletion associated with lambda-gene rearrangements in various cell lines. All V and C gene segments were found to be in the same transcriptional orientation, and V2 and V1 were found not to be next to each other, thus supporting the gene order V2-C2-C4-V1-C3-C1.