Genetic and biochemical analysis of CodY-binding sites in Bacillus subtilis.
CodY is a global transcriptional regulator that is known to control directly the expression of at least two dozen operons in Bacillus subtilis, but the rules that govern the binding of CodY to its target DNA have been unclear. Using DNase I footprinting experiments, we identified CodY-binding sites upstream of the B. subtilis ylmA and yurP genes. The protected regions overlapped versions of a previously proposed CodY-binding consensus motif, AATTTTCWGAAAATT. Multiple single mutations were introduced into the CodY-binding sites of the ylmA, yurP, dppA, and ilvB genes. The mutations affected both the affinity of CodY for its binding sites in vitro and the expression in vivo of lacZ fusions that carry these mutations in their promoter regions. Our results show that versions of the AATTTTCWGAAAATT motif, first identified for Lactococcus lactis CodY, with up to five mismatches play an important role in the interaction of B. subtilis CodY with DNA.
Belitsky
Boris R
BR
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA. Boris.Belitsky@tufts.edu
Sonenshein
Abraham L
AL
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000494:pharmacology; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins / Q000235:genetics*; D024363:Transcription Initiation Site
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D006160:Guanosine Triphosphate