title: Operator interactions by the Bacillus subtilis arginine repressor/activator, AhrC: novel positioning and DNA-mediated assembly of a transcriptional activator at catabolic sites.
abstract: We have previously reported the initial characterization of a catabolic operator site (O[rocA]) for the Bacillus subtilis arginine repressor/activator protein AhrC. Here, we present the characterization by gel retardation and DNase I footprinting of both O(rocA) and a second catabolic operator site, O(rocD). Both operator sites encompass a single recognition site, an ARG box, located immediately upstream of the transcriptional start points, a unique positioning for a transcriptional activator protein. Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around twofold, in their apparent affinities for the protein (K'd approximately 90 nM for O[rocA] and approximtaely 190nM for O[rocD]). This difference may result from the lower match to the ARG box consensus of the O(rocD) site. Both catabolic operators show evidence for co-operative binding with respect to protein concentration. Determination of the sequences of two AhrC catabolic operator sites, in combination with the three such biosynthetic sites, has allowed the derivation of an improved B. subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t. This is not identical to the Escherichia coli consensus operator for the AhrC homologue, ArgR, which may explain the only partial cross-functioning of these proteins in vivo. The O(rocA) site is adjacent to a sharp, stable bend located 5' to the catabolic operator. Circular permutation analysis has been used to determine the relative angle of bend (approximately 50 degrees), its location and the effect of adding magnesium ions and/or AhrC protein. Protein binding increases the relative bend angle to approximately 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. Very surprisingly, the affinity of the O(rocA) site appears to increase in the presence of excess, specific competitor fragment, i.e. the system shows anti-competitive effects. Competition is restored at high molar excesses of specific fragment over the protein. We propose a novel model for the assembly of a higher affinity form of AhrC at operator sites that is consistent with both the apparent co-operativity of binding and the anti-competitive effects. These data suggest that the molecular interactions occurring between the prokaryotic arginine-regulatory proteins and their operators may be more complex than is generally appreciated.
authors.affiliation: School of Biology, University of Leeds, UK.
authors.forename: C M
authors.initials: CM
authors.lastname: Miller
authors.forename: S
authors.initials: S
authors.lastname: Baumberg
authors.forename: P G
authors.initials: PG
authors.lastname: Stockley
mesh_terms: D001120:Arginine / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D009875:Operator Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: C087018:ArgR protein, Bacteria; C490177:ArgR protein, E coli; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D001120:Arginine