In a previous publication (He, J.-S., and Fulco, A. J.(1991) J. Biol. Chem. 266, 7864-7869), we reported that a 15-17-base pair DNA sequence (designated a Barbie box element) in the 5`-regulatory regions of cytochrome P450and P450 genes from Bacillus megaterium was recognized by a barbiturate-regulated protein. It is now recognized that essentially all eukaryotic and prokaryotic genes whose 5`-flanking regions are known and that encode barbiturate-inducible proteins contain the Barbie box element. A 4-base pair sequence (AAAG) is found in the same relative position in all Barbie box elements. In B. megaterium, mutation of the Barbie box located in the P450 gene leads to the constitutive synthesis of cytochrome P450 and a 10-fold increase of expression of Bm1P1, a small gene located upstream of the P450 gene, that encodes a putative regulatory protein. Mutation of the P450 Barbie box significantly increased the expression of both P450 and Bm3P1 (another small gene located upstream of the P450 gene that encodes a second putative regulatory protein) in response to pentobarbital induction but left the basal levels unaffected. In gel mobility shift assays, Bm3R1, a repressor of the P450 gene, was found to specifically interact with the Barbie box sequences of the B. megaterium P450 genes. Mutated Barbie boxes showed a decreased binding affinity for Bm3R1 compared to their wild type (unmutated) counterparts. Barbie box sequences were also shown to specifically interact with putative positive regulatory factors of B. megaterium cells. These putative positive factors were induced by pentobarbital and were also present at high levels during late stationary phase of B. megaterium cell cultures grown in the absence of barbiturates. The mutated Barbie box sequences had greater binding affinity for these positive factors than did unmutated Barbie box sequences. DNase I footprinting analysis of the 5`-flanking region of the P450 gene revealed that these positive factors protected a segment of DNA covering a portion of the Barbie box sequence and a small flanking region. Similar footprinting experiments with the 5`-flanking region of the P450 gene failed, however, to unambiguously reveal protected sequences in the Barbie box region. The evidence suggests that the positive factors and Bm3R1 compete with each other for binding to the Barbie box region, especially in the 5`-flanking region of the P450 gene, and for putative roles in the regulation of transcription from the B. megaterium P450 genes. This inference was further supported by evidence derived from a nested-deletion analysis in and around the Barbie box of the P450 regulatory region that showed that a repressor binding site and a positive factor binding site overlap at the Barbie box sequence. In toto, these experimental results indicate that, in B. megaterium at least, the Barbie box sequences are important cis-acting elements for coordinately regulating the barbiturate-mediated expression of the P450 and P450 genes and the genes encoding their positive regulatory factors.

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