STOICHIOMETRY OF BINDING OF CYSB TO THE CYSJIH CYSK, AND CYSP PROMOTER REGIONS OF SALMONELLA-TYPHIMURIUM

CysB is a member of the LysR family of transcriptional activators and regulates genes of the cysteine regulon in Salmonella typhimurium and Escherichia coli. CysB binds to specific sites just upstream of the -3 5 regions of the cysJIH, cysK, and cysP promoters, where, in the prese nce of N-acetyl-L-serine, it stimulates transcription initiation. The cysK and cysP promoters contain additional binding sites, and we have proposed that CysB bends these promoters by binding to adjacent sites. N-Acetyl-L-serine is thought to decrease the magnitude of such bendin g. Since stoichiometric data bearing on this model have been lacking, we analyzed complexes in gel mobility shift experiments with S-35-Iabe led CysB and P-32-labeled promoter fragnients. CysB was found to bind as a tetramer, and N-acetyl-L-serine increased the electrophoretic mob ilities of one-protein complexes of the multibinding site cysK and cys P promoters without changing their stoichiometry, indicating that a si ngle CysB tetramer can bend these promoters and that N-acetyl-L-serine diminishes such bending. Bend angles for both promoters were calculat ed to be 100 and 50 degrees in the absence and presence of N-acetyl-L- serine. N-Acetyl-L-serine affected neither the stoichiometry nor the e lectrophoretic mobility of cysJIH promoter complexes, which are not kn own to contain bent DNA. DNA bending may be a mechanism for sequesteri ng CysB at certain promoter sites by increasing their affinity for thi s protein in the absence of N-acetyl-L-serine.