DNA Looping and Unlooping by AraC Protein

Abstract

Expression of the L-arabinose BAD operon in Escherichia coli is regulated by AraC protein which acts both positively in the presence of arabinose to induce transcription and negatively in the absence of arabinose to repress transcription. The repression of the araBAD promoter is mediated by DNA looping between AraC protein bound at two sites near the promoter separated by 210 base pairs, araI and araO2. In vivo and in vitro experiments presented here show that an AraC dimer, with binding to half of araI and to araO2, maintains the repressed state of the operon. The addition of arabinose, which induces the operon, breaks the loop, and shifts the interactions from the distal araO2 site to the previously unoccupied half of the araI site. The conversion between the two states does not require additional binding of AraC protein and appears to be driven largely by properties of the protein rather than being specified by the slightly different DNA sequences of the binding sites. Slight reorientation of the subunits of AraC could specify looping or unlooping by the protein. Such a mechanism could account for regulation of DNA looping in other systems.

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

“…Members of the family for which in vitro or in vivo footprinting assays are available (AraC, RhaR, RhaS, MelR, SoxS, and Ada) have at least two features in common: they function in vivo as a dimer, and the stretch of nucleotides covered by a monomer of the regulatory protein at the regulated promoter is between 15 and 20 bp long. However, within this set of bases, short motifs seem to confer critical base recognition for DNA-protein interactions (15)(16)(17)(18)(19)(20)(21). This seems also to be the case for Pm/XylS interactions.…”

The role of rigidity in DNA looping-unlooping by AraC

“…Another property that can be attributed to rigidity can be seen in the binding of AraC in the absence of arabinose to two adjacent I 1 and I 2 half-sites. Whereas such binding normally does not occur in vivo, it can be observed in vitro (3,6). According to the current view, to bind to adjacent half-sites, part of the DNA binding energy must be used to distort AraC so that the DNA binding domains are correctly positioned adjacent to one another.…”

“…1. On the addition of arabinose, the protein's affinity for the I 1 and I 2 half-sites increases by about 50-fold, leading the protein to prefer to bind to these adjacent half-sites and induce the p BAD promoter rather than loop and repress p BAD (3,(5)(6)(7)(8). The basis for the change in the DNA binding properties appears to result from an arabinoseinduced shift of the N-terminal arms from associating with the DNA-binding domains of AraC to associating with the dimerization domains (refs.…”

“…ata obtained from both in vivo and in vitro experiments have shown that, in the absence of arabinose, the dimeric AraC protein prefers binding to the well separated I 1 and O 2 half-sites and forming a DNA loop (1)(2)(3)(4), Fig. 1.…”

Pheromone-inducible conjugation in Enterococcus faecalis: A model for the evolution of biological complexity?

“…Interestingly, PrgX was found to require the DNA loop in order to regulate its own production and that of Qa RNA; indicating that the DNA loop regulates transcription from both strands of pCF10 DNA. There are many examples of regulatory proteins that utilize DNA loop formation for control of multiple promoters (Anderson et al, 1981;Lobell and Schlief, 1990). PrgX, however is the first example to our knowledge of a repressor that simultaneously controls transcripts produced from both strands of its target DNA loop by acting on one promoter, while participating in a post-transcriptional event in the opposite direction.…”