Purification and characterization of the DeoR repressor of Bacillus subtilis.
Transcription of the Bacillus subtilis dra-nupC-pdp operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the dra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.
Zeng
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Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark.
Saxild
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HH
Switzer
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RL
D000494:Allosteric Regulation / Q000187:drug effects; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D007700:Kinetics; D008970:Molecular Weight; D009875:Operator Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000378:metabolism / Q000494:pharmacology; D013816:Thermodynamics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C489070:DeoR protein, E coli; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012267:Ribosemonophosphates; C058245:2-deoxyribose 5-phosphate; D004247:DNA