A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome.
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.
Kitagawa
R
R
Department of Molecular Biology, School of Science, Nagoya University, Japan.
Mitsuki
H
H
Okazaki
T
T
Ogawa
T
T
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D012097:Repressor Proteins