Alternative DNA loops regulate the arabinose operon in Escherichia coli.

Abstract

The araCBAD regulatory region of Escherichia coli contains two divergently oriented promoters and three sites to which AraC, the regulatory protein of the operon, can bind. This paper presents the results of in vivo dimethyl sulfate "footprinting" experiments to monitor occupancy of the three AraC sites and measurements of activity of the two promoters. These measurements were made both in the absence of the inducer arabinose and at various times after arabinose addition to growing cells containing the wild-type ara regulatory region or the regulatory region containing various deletions and point mutations. The data lead to the conclusion that two different DNA loops can form in the ara regulatory region. These loops are generated by AraC protein molecules binding to two different DNA sites and binding to each other. One of these loops predominates in the absence of arabinose and plays a major role in repressing activity of one of the promoters. Upon the addition of arabinose the amount of the first loop type, the repression loop, decreases and the amount of a second loop increases. Formation of this second loop precludes the counterproductive formation of the repression loop.AraC protein is a multifunctional regulatory protein of the genes required for uptake and catabolism of L-arabinose in Escherichia coli (1)(2)(3)(4). This paper concerns the inducing and repressing activities of AraC on the two promoters located in the regulatory region between the araC and araBAD genes. One, PBAD, serves the araBAD catabolic genes, and the other, Pc, serves the araC regulatory gene. In the presence of arabinose, AraC protein bound at the araI site, which is immediately adjacent to the RNA polymerase binding site of the PBAD promoter, stimulates transcription of the araBAD genes (Fig. 1) (8,9).A variety of experiments have led to the conclusion that looping occurs in the ara system (5-7, 10). Additionally, DNA looping appears to be a common mechanism for gene regulation, as a number of other systems also show one or more of the properties displayed by the ara system and can be interpreted to loop so that two proteins bound to separated sites on DNA are in direct contact (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Finally, the strength of the protein-DNA interactions and the proteinprotein interactions in some systems (14,16,20,22) permits loops generated on linear DNA to be observed in the electron microscope.Previous in vivo dimethyl sulfate methylation "footprinting" studies of the araCBAD operon revealed that in the absence of the two other AraC protein binding sites, araO2 binds AraC protein so weakly that it is virtually unoccupied (9). That is, the presence of the other sites, even though they are located >100 bp away, greatly increases AraC protein occupancy of araO2. Since AraC bound at araO2 must be interacting with one or both of the other sites to generate this binding-site cooperativity, either they are communicating by sending a signal along the DNA between the sites or, more likely, the DNA is lo...

Negative regulatory loci coupling flagellin synthesis to flagellar assembly in Salmonella typhimurium

“…We propose that this looping of the DNA resulting from Hin tetramer formation plays a role in the switching bias. Long-range protein-protein interactions have been shown to be involved in regulation of the ara, deo, gal, and lac operons of E. coli (9,11,14,15,20,48). In vivo binding of Hin to a defective hix site is enhanced by the presence of another functional hix site even at a distance of 1 kb (16a), suggesting a role of tetramer formation in binding.…”

Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription.

“…The sequence TCGAAG happened to be identical to the -35 sequence of deoP2 promoter (6). The three -10 sequences include GAGAAT from the OmpRdependent ompC promoter (8,9), TACTGT from the AraCdependent araBAD promoter (6,14), and TATACT from the constitutive Ipp P-5 promoter (16). The last -10 sequence has been shown to exert stronger transcriptional activities than the consensus -10 sequence (TATAAT; ref.…”

“…1B) were used. Of the nine -35 sequences, the following were used: TTGACA for the consensus sequence (6), TAGCAG from ompF, TTGGAT from ompC (7-9), TTTAAG from the MalT-dependent malK promoter (4, 6), CTGACG from the AraC-dependent araBAD promoter (6,14), and CTCACT from cAMP receptor proteindependent lacP2 promoters (6,15) TCGAAG, TCGTCC, and ATCACA. The last three sequences were designed to generate three mismatches at different positions within the consensus sequences.…”

Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon

“…The fragments were filled out with DNA polymerase (Klenow fragment) and then ligated into the SmaI site in the polylinker region of pUC19. The 1.9-kb clone was digested at the EcoRI and HindIll sites present on the polylinker and cloned into pES51 (15), which contains the M13 origin of replication. To speed sequencing, the 2.5-kb clone was further divided by cutting at an internal EcoRI site as well as the flanking EcoRI site in the polylinker; the resulting 1.0-and 1.5-kb fragments were then ligated into the EcoRI site in pES51.…”