OPERATOR INTERACTIONS BY THE BACILLUS-SUBTILIS ARGININE REPRESSOR ACTIVATOR, AHRC - NOVEL POSITIONING AND DNA-MEDIATED ASSEMBLY OF A TRANSCRIPTIONAL ACTIVATOR AT CATABOLIC SITES/

We have previously reported the initial characterization of a cataboli c operator site (O-rocA) for the Bacillus subtilis arginine repressor/ activator protein AhrC. Here, we present the characterization by gel r etardation and DNase I footprinting of both O-rocA and a second catabo lic operator site, O-rocD. Both operator sites encompass a single reco gnition site, an ARG box, located immediately upstream of the transcri ptional start points, a unique positioning for a transcriptional activ ator protein. Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around two fold, in their apparent affinities for the protein (K-d' approximate t o 90 nM for O-rocA and approximate to 190 nM for O-rocD). This differe nce may result from the lower match to the ARG box consensus of the O- rocD site. Both catabolic operators show evidence for co-operative bin ding with respect to protein concentration. Determination of the seque nces of two AhrC catabolic operator sites, in combination with the thr ee such biosynthetic sites, has allowed the derivation of an improved B. subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t. This i s not identical to the Escherichia coil consensus operator for the Ahr C homologue, ArgR, which may explain the only partial cross-functionin g of these proteins in vivo. The O-rocA site is adjacent to a sharp, s table bend located 5' to the catabolic operator. Circular permutation analysis has been used to determine the relative angle of bend (approx imate to 50 degrees), its location and the effect of adding magnesium ions and/or AhrC protein. Protein binding increases the relative bend angle to approximate to 85 degrees. Bending is shown to be associated with a number of A-tracts in the upstream sequence. However, altering the phasing of the A-tracts has little effect on the affinity for AhrC . Truncation and competition experiments have been used to investigate the possible role of sequences flanking the operator on affinity. Ver y surprisingly, the affinity of the O-rocA site appears to increase in the presence of excess, specific competitor fragment, i.e. the system shows anticompetitive effects. Competition is restored at high molar excesses of specific fragment over the protein. We propose a novel mod el for the assembly of a higher affinity form of AhrC at operator site s that is consistent with both the apparent co-operativity of binding and the anti-competitive effects. These data suggest that the molecula r interactions occurring between the prokaryotic arginine-regulatory p roteins and their operators may be more complex than is generally appr eciated.