Specific binding of DnaA protein to a DnaA box in the guaB gene of Escherichia coli K12

Expression of the guaBA operon of Escherichia coli is regulated by the DNA replication-initiating protein, DnaA. Two DnaA boxes, which are potential binding sites for DnaA, are present in the gun operon. One box (with 8/9 match to the DnaA box consensus sequence) is at the gua promoter; the other box, which has a consensus sequence, is on the non-transcribed strand within the guaB coding region approximately 200 bp downstream of the initiation codon. The binding in vitro of purified DnaA protein to these boxes was investigated by filter retention and gel retardation analysis, and by deoxyribonuclease I footprinting, using restriction fragments of gun operon DNA. DnaA protein was shown to bind specifically only to the fragment carrying the consensus sequence DnaA box, and to protect this box from deoxyribonuclease I. Transcription termination resulting from the binding of DnaA to this box within the guaB gene explains repression by DnaA of the gun operon in vivo.