In vitro selection of optimal AbrB-binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures.

Abstract

The AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters. Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination. Using in vitro selection techniques, we have isolated optimal AbrB-binding sites from oligonucleotides containing 22 or 44 random base pairs. The best of these sites have an apparent in vitro Kd which is fivefold lower than a similar-sized DNA fragment containing the sequence corresponding to the AbrB-binding site on the spo0E gene. We tested one of the sites in vivo and found that it confers AbrB-mediated control upon a promoter not normally regulated by AbrB. In each of four separate trials, the selected sites possess motifs that converge to a simple consensus. It is argued that the nature and spacing of these motifs produce a type of three-dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant.