| Abstract |
There is a DNA sequence which has unusually high affinity for the DnaA protein of Escherichia coli between the glyV and amiB-mutL operons at 94.7 min on the genetic map. Affinity of DnaA protein for DNA was measured in vivo as the activity of beta-galactosidase produced from the lacZ gene on a plasmid fused to the 5'-terminal portion of the mioC gene, which is under the control of the DnaA protein. The chromosomal DNA segment between the two operons, carried on a compatible plasmid, derepressed the beta-galactosidase activity by titrating DnaA protein. Derepression occurred on the chromosomal dnaA gene as well, since it is autoregulated. Hence, as measured by immunoassays, one plasmid molecule carrying the DnaA-binding region titrated 370 dnaA molecules, which is a value eightfold higher than that for a plasmid containing the oriC region. We estimate that about 60% of the total cellular DnaA molecules are bound to this site. Four DnaA-binding sequences (DnaA boxes) and a DnaA-regulated promoter directing transcription of two small genes were present within a 250 bp stretch in this region but additional long DNA regions, including the fifth DnaA box located about 650 bp downstream, were required for maximum binding. A role for the DnaA-binding site in controlling DnaA-protein concentration in the cell cycle is discussed.
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