The presence of two DnaA-binding sequences is required for an efficient interaction of the Escherichia coli DnaA protein with each particular weak DnaA box region

Using an electron microscopic method for visualizing interactions of the Es cheridia coli DnaA protein with weak DnaA-binding DNA sequences, we found t hat DnaA binds effectively to two separated weak DnaA box regions located o n the same DNA fragment. As expected, no DnaA-DNA interactions were detecte d when both DnaA box regions were mutagenized to the sequence totally incap able of binding DnaA. However, when only one of these two regions was mutag enized, the lack of interactions between DnaA and DNA was observed not only at the scrambled DnaA box but also at the second weak DnaA box region. The se results indicate that for the efficient binding of DnaA to a weak DnaA b ox region, the presence of at least two such DNA sequences is necessary. Ou r finding also suggests that binding of DnaA protein to weak DnaA box seque nces may be cooperative. In addition, we found that DnaA-mediated transcrip tion termination in vivo requires two DnaA boxes, one of them is a weak one . It seems, therefore, that some mechanisms of regulation of transcription and DNA replication by DnaA, that involve interactions of DnaA with weak Dn aA boxes, may be more complicated than initially proposed. (C) 1999 Federat ion of European Microbiological Societies. Published by Elsevier Science B. V. All rights reserved.