DNA-Binding properties of the Fremyella diplosiphon RpbA repressor.
Mutant strain FdBM1 of the cyanobacterium Fremyella diplosiphon is characterized by elevated transcription of the cpcB1A1 gene set due to inactivation of rpbA by Tn5469. The predicted RpbA protein contains two regions resembling the characterized helix-turn-helix (HTH) motif involved in DNA recognition by many phage and bacterial transcription regulator proteins. It was therefore hypothesized that RpbA functions as a DNA-binding repressor involved in the control of transcription from cpcB1A1. A histidine-tagged form of RpbA, designated RpbA-His(6), was examined for its ability to bind to the defined promoter region for cpcB1A1. Gel mobility shift assays showed that RpbA-His(6) specifically binds to a DNA fragment containing the cpcB1A1 promoter and that significant binding can be achieved with equimolar amounts of RpbA-His(6) and the cpcB1A1 promoter probe. DNase I footprint analysis localized the RpbA-His(6) binding site to an asymmetric 21-bp region that overlaps the putative -10 promoter sequence. A mutational analysis suggested that binding by RpbA-His(6) to its cognate DNA may involve both putative HTH motif-like regions. We conclude that RpbA functions as a transcriptional repressor for cpcB1A1 and suggest that binding by RpbA to its cognate DNA may represent an atypical protein-DNA interaction.
Manna
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Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.
Nieder
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Schaefer
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D019077:Helix-Turn-Helix Motifs; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010798:Phycocyanin / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D010798:Phycocyanin; D006639:Histidine