The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli.
After binding to its four 9-mer boxes in the 245-base pair Escherichia coli replication origin (oriC), dnaA protein effects the formation of an "open complex" in an adjacent region made up of three 13-mers (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). This open complex formation requires the ATP form of dnaA protein assisted by HU protein (Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50, 259-265). We now provide direct evidence that dnaA protein binds the 13-mers, sequences that bear no resemblance to the 9-mer box. The evidence is (i) displacement of dnaA protein from the open complex by oriC or by a synthetic oligonucleotide containing the 13-mers, but not by a mutant of oriC lacking the 13-mers; (ii) filter binding of the synthetic (13-mer) oligonucleotide by dnaA protein; and (iii) requirement for the ATP form of dnaA protein assisted by HU protein for temperature-dependent binding to the 13-mer region. Controlled proteolysis of dnaA protein results in a prompt loss of oriC binding; an NH2-terminal 30-kDa peptide contains the domain that binds ATP and phospholipids known to destabilize the tightly bound ATP.
Yung
B Y
BY
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307.
Kornberg
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin