Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the lambda DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

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It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.

Using an electron microscopic method for visualizing interactions of the Escherichia coli DnaA protein with weak DnaA-binding DNA sequences, we found that DnaA binds effectively to two separated weak DnaA box regions located on the same DNA fragment. As expected, no DnaA-DNA interactions were detected when both DnaA box regions were mutagenized to the sequence totally incapable of binding DnaA. However, when only one of these two regions was mutagenized, 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. These results indicate that for the efficient binding of DnaA to a weak DnaA box region, the presence of at least two such DNA sequences is necessary. Our finding also suggests that binding of DnaA protein to weak DnaA box sequences may be cooperative. In addition, we found that DnaA-mediated transcription 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 DnaA boxes, may be more complicated than initially proposed.

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