Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control.
CysB is a tetrameric LysR-type transcriptional regulator that acts as an activator of cys regulon genes and as an autorepressor. Positive control of cys genes requires the presence of the inducer N-acetylserine. Following random and site-directed mutagenesis of the cysB gene, 20 CysB variants were isolated. Six single amino acid substitutions within the N terminus of CysB abolished the DNA-binding ability of the protein. Seven mutations in the central region of CysB affected its response to the inducer. Four of these CysB mutants retained repressing activity, but lost their activating function in vivo. Their DNA binding characteristics were consistent with an inability to respond to acetylserine by a qualitative change in the DNA-protein interaction. Three of the single residue substitutions resulted in constitutive activity of CysB. The electrophoretic mobility of the complex formed by one of the CysBc variants with the cysP promoter suggested a dimeric state of this protein. Characteristics of six truncated CysB variants lacking 5-30 C-terminal residues indicated the involvement of the C terminus in the DNA binding, oligomerization, and stability of CysB. The single substitution Y27G resulted in the CysBpc variant, able to bind DNA and to respond to the inducer by a qualitative change in the DNA-protein complex, but defective in the positive control of the cysP promoter.
Lochowska
A
A
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
Iwanicka-Nowicka
R
R
Plochocka
D
D
Hryniewicz
M M
MM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D010641:Phenotype; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D014157:Transcription Factors; C073470:LysR protein, Bacteria; D012321:DNA-Directed RNA Polymerases