DNA bending by AraC: A negative mutant

Abstract

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. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild- type AraC bends DNA about 90°whereas the mutant bends DNA by a smaller amount.