THE DNA-BINDING DOMAIN OF THE INITIATOR PROTEIN DNAA

The 94 C-terminal amino acids of the initiator protein DnaA of Escheri chia coil are required and sufficient for specific binding to the cogn ate DNA binding site, The binding domain contains two potential amphip athic alpha-helices and a third alpha-helix, It represents a new DNA b inding motif so far not found in other DNA binding proteins, Temperatu re-sensitive mutations in the binding motif, dnaA204, dnaA205 and dnaA 211, abolish DNA binding, In the solid-phase DNA binding assay, applic able to other DNA binding proteins, fusions of domains of DnaA protein to beta-galactosidase are reacted with biotinylated anti-beta-galacto sidase antibody, These are coupled to streptavidin-coated magnetic bea ds, The DNA binding domain is able to selectively remove the DNA targe t (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 dir ectly to streptavidin-coated magnetic beads, Homologies between DnaA p rotein and transcription factors of the NtrC family are discussed.