IN-VITRO SELECTION OF OPTIMAL ABRB-BINDING SITES - COMPARISON TO KNOWN IN-VIVO SITES INDICATES FLEXIBILITY IN ABRB BINDING AND RECOGNITION OF 3-DIMENSIONAL DNA STRUCTURES

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 analyse d by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination. Using in vitro selection technique s, we have isolated optimal AbrB-binding sites from oligonucleotides c ontaining 22 or 44 random base pairs. The best of these sites have an apparent in vitro K-d which is fivefold lower than a similar-sized DNA fragment containing the sequence corresponding to the AbrB-binding si te on the spo0E gene. We tested one of the sites in vivo and found tha t it confers AbrB-mediated control upon a promoter not normally regula ted by AbrB. In each of four separate trials, the selected sites posse ss motifs that converge to a simple consensus. It is argued that the n ature and spacing of these motifs produce a type of three-dimensional DNA structure recognizable by AbrB, and that known in vivo sites, whic h lack these motifs, possess an approximation of the optimal structura l determinant.