LOCATING AND LENGTHENING THE INTERDOMAIN LINKER IN ARAC PROTEIN

A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions. Attractive candidates for th ese areas are linker regions. Such a region was found to include posit ions 171 to 178 in the Escherichia coli regulatory protein AraC. Indep endent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domai n. Hence, by both the genetic and biochemical approaches, the interdom ain linking region was determined to include amino acid residues 171 t o 177. The properties of altered proteins were examined using template s with AraC half-sites more widely separated than in the wild-type cas e. Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerizatio n and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site. In vitro, the pr oteins with longer linkers bound substantially more stably than wild-t ype AraC to the DNA containing half-sites for AraC separated by an ext ra two helical turns of DNA. In vivo on an ara promoter with the more widely separated AraC half-sites, the proteins could activate transcri ption much better than wild-type AraC.