title: Burkholderia xenovorans RcoM(Bx)-1, a transcriptional regulator system for sensing low and persistent levels of carbon monoxide.
abstract: The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal "LytTR" DNA-binding domain. Here the RcoM(Bx)-1 protein from Burkholderia xenovorans LB400 was heterologously expressed and then purified in a form with minimal bound CO (~10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ~60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5'-TTnnnG-3') that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms. In vivo screens confirmed the functional importance of the conserved "TTnnnG" motif residues and their triplet arrangement and were also used to determine an improved binding motif [5'-CnnC(C/A)(G/A)TTCAnG-3'] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoM(Bx)-1 to a variety of DNA probes was demonstrated in vitro. We posit that for the RcoM(Bx)-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional "accumulating switch" that senses low but persistent CO levels.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA. groberts@bact.wisc.edu
authors.forename: Robert L
authors.initials: RL
authors.lastname: Kerby
authors.forename: Gary P
authors.initials: GP
authors.lastname: Roberts
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019117:Burkholderia / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism*; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014157:Transcription Factors; D002248:Carbon Monoxide