The DNA binding domain of the initiator protein DnaA.
The 94 C-terminal amino acids of the initiator protein DnaA of Escherichia coli are required and sufficient for specific binding to the cognate DNA binding site. The binding domain contains two potential amphipathic alpha-helices and a third alpha-helix. It represents a new DNA binding motif so far not found in other DNA binding proteins. Temperature-sensitive mutations in the binding motif, dnaA204, dnaA205 and dnaA211, abolish DNA binding. In the solid-phase DNA binding assay, applicable to other DNA binding proteins, fusions of domains of DnaA protein to beta-galactosidase are reacted with biotinylated anti-beta-galactosidase antibody. These are coupled to streptavidin-coated magnetic beads. The DNA binding domain is able to selectively remove the DNA target (oriC) from the liquid phase. Alternatively, the DNA binding domain is fused to a peptide containing a target sequence which is naturally biotinylated in vivo in E.coli. This fusion protein can be coupled directly to streptavidin-coated magnetic beads. Homologies between DnaA protein and transcription factors of the NtrC family are discussed.
Roth
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Max-Planck-Institut für Molekulare Genetik, Berlin-Dahlem, Germany.
Messer
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D051922:PII Nitrogen Regulatory Proteins; D017433:Protein Structure, Secondary; D013696:Temperature; D015534:Trans-Activators; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli