Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientationand spatial relationship to the core promoter

The promoters of the mar/sox/rob regulon of Escherichia coli: contain a bin ding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not be en precisely defined because of its degeneracy and asymmetry and seemingly variable location with respect to the -10 and -35 hexamers for RNA polymera se (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of prom oters. This has yielded a more defined marbox consensus sequence (AYnG-CACn nWnnRYYAAAYn) and has led to the demonstration that some marboxes are inver ted relative to others. Using transcriptional fusions to lacZ, we have foun d that only one marbox orientation is functional ata given location, Moreov er, the functional orientation is determined by marbox location: marboxes t hat are 15 or more basepairs upstream of the -35 hexamer are oriented oppos ite those closer to the -35 hexamer. Marbox orientation and the spacing bet ween marbox and signals for RNP binding are critical for transcriptional ac tivation, presumably to align MarA with RNP.