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 gua operon. One box (with 819 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 gua 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 gua operon in vivo.Keywords: guaBA operon ; transcriptional control ; DnaA protein; Escherichia coli.In Escherichia coli, bidirectional DNA replication is initiated at a distinct region of the chromosome known as oriC (origin of chromosome replication). DnaA protein, the product of the dnaA gene, is essential for initiation of replication from oriC both in vivo [l] and in vitro [2]. DnaA binds to oriC specifically and cooperatively at 9-bp repeats (DnaA boxes of consensus sequence TTAT(C,)CA(C,)A [3]) present four times within the minimal origin region of 245 bp. After binding, DnaA organizes the assembly of the replisome complex by directing other proteins required for initiation and replication to this site [4, 51.In addition to being required for replication initiation, DnaA also acts as a repressor and/or terminator of transcription for several genes. One of the genes affected is the dnaA gene itself 16, 71. Other genes for which there is direct or circumstantial evidence for regulation by DnaA are mioC [8] [12], pros (drpA) [13], and nrdAB [14]. The products of most of these genes are concerned with the major macromolecular processes of replication and modification of DNA, transcription and translation. The dnaA gene is autoregulated by the binding of DnaA to a DnaA box located between its two promoters [6,[15][16][17]. Messer et al. [7] proposed that when DnaA binds to a second DnaA box, within the dnaAcoding region, it acts as a transcriptional terminator. Termination of transcription was shown to be orientation dependent [18]; a DnaA box located on the non-transcribed strand, which is orientated in the direction of transcription, is necessary for transcription termination.We have recently reported [19] that the guaBA operon, which is responsible for the production of the final two enzymes for guanine nucleotide biosynthesis, is regulated by DnaA protein. When the intracellular concentration of DnaA is increased (by induction of a multicopy plasmid carrying the dnaA gene fused to a tac promoter), expression of the gua operon is reduced. Decreasing the intracellular...
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