Purification of a MalE-SoxS fusion protein and identification of the control sites of Escherichia coli superoxide-inducible genes.
In Escherichia coli, the soxRS genes effect the cell's defence against superoxide by activating the transcription of more than 14 genes, including zwf, sodA, nfo, micF and fumC. Previous work from other laboratories has indicated that SoxR is the sensor of oxidative stress and induces synthesis of SoxS, which in turn activates transcription of the regulon's target genes. Although SoxS appears to be a DNA-binding protein, its ability to bind to the promoter regions of target genes has not been demonstrated. To facilitate purification and characterization of SoxS, we constructed a fusion of soxS to malE, which encodes maltose-binding protein, and demonstrated that the in vivo expression of the MalE-SoxS fusion protein can provide SoxS function to a soxRS deletion mutant. We purified the fusion protein by affinity chromatography on an amylose column. The purified fusion protein stimulated in vitro expression of zwf in a coupled transcription-translation system and formed specific complexes with DNA fragments carrying target gene promoters. Moreover, MalE-SoxS protected from DNase I attack 22-27 bp segments immediately adjacent to or overlapping the -35 hexamers of the zwf, sodA, nfo, micF, and fumC promoters. The protected regions revealed a consensus 'soxbox' sequence.
Fawcett
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WP
Department of Biological Sciences, University of Maryland, Catonsville 21228.
Wolf
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D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D018384:Oxidative Stress; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C461599:maltose transport system, E coli; D013481:Superoxides; C070725:SoxS protein, E coli; D003850:Deoxyribonuclease I