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 -35 regions of the cysJIH, cysK, and cysP promoters, where, in the presence 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 bending. Since stoichiometric data bearing on this model have been lacking, we analyzed complexes in gel mobility shift experiments with 35S-labeled CysB and 32P-labeled promoter fragments. CysB was found to bind as a tetramer, and N-acetyl-L-serine increased the electrophoretic mobilities of one-protein complexes of the multibinding site cysK and cysP promoters without changing their stoichiometry, indicating that a single CysB tetramer can bend these promoters and that N-acetyl-L-serine diminishes such bending. Bend angles for both promoters were calculated 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 electrophoretic mobility of cysJIH promoter complexes, which are not known to contain bent DNA. DNA bending may be a mechanism for sequestering CysB at certain promoter sites by increasing their affinity for this protein in the absence of N-acetyl-L-serine.
Hryniewicz
M M
MM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Kredich
N M
NM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D003545:Cysteine / Q000096:biosynthesis; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics*; D008957:Models, Genetic; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D018085:Regulon / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C062886:CysB protein, Bacteria; C489265:CysB protein, Salmonella typhimurium; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D012694:Serine; C073470:LysR protein, Bacteria; D003545:Cysteine; C012162:N-acetylserine