DNA bending by AraC: a negative mutant.

We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of Escherichia coli that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were trans dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the araBAD promoter was studied further.

Arm-domain interactions in AraC.

N-terminal deletions extending beyond the sixth amino acid of the Escherichia coli regulator of the l-arabinose operon, AraC, were found to generate constitutive regulatory behavior of the promoter pBAD. Mutagenesis of the DNA coding for the first 20 amino acids of the protein and screening for constitutives yielded mutants across the region whereas screening for mutants that cannot induce pBAD, even in the presence of arabinose, yielded none. These results indicate that the N-terminal arm is not essential for transcription activation, but that it plays an important and active role in holding the system in a non-activating state.

Apo-AraC actively seeks to loop.

In the absence of arabinose and interactions with other proteins, AraC, the activator-repressor that regulates the araBAD operon in Escherichia coli, was found to prefer participating in DNA looping interactions between the two well-separated DNA half-sites, araI1 and araO2 at their normal separation of 211 base-pairs rather than binding to these same two half-sites when they are adjacent to one another. On the addition of arabinose, AraC preferred to bind to the adjacently located half-sites. Inverting the distally located araO2 half-site eliminated the looping preference.