THE ROLE OF BARBIE BOX SEQUENCES AS CIS-ACTING ELEMENTS INVOLVED IN THE BARBITURATE-MEDIATED INDUCTION OF CYTOCHROMES P45(BM-1) AND P450(BM-3) IN BACILLUS-MEGATERIUM

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 sequenc e (designated a Barbie box element) in the 5'-regulatory regions of cy tochrome P450(BM-1) and P450(BM-3) genes from Bacillus megaterium was recognized by a barbiturate-regulated protein. It is now recognized th at essentially all eukaryotic and prokaryotic genes whose 5'-flanking regions are known and that encode barbiturate-inducible proteins conta in the Barbie box element, A 4-base pair sequence (AAAG) is found in t he same relative position in all Barbie box elements, In B. megaterium , mutation of the Barbie box located in the P450(BM-1) gene leads to t he constitutive synthesis of cytochrome P450(BM-1) and a 10-fold incre ase of expression of Bm1P1, a small gene located upstream of the P450( BM-1) gene, that encodes a putative regulatory protein, Mutation of th e P450(BM-3) Barbie box significantly increased the expression of both P450(BM-3) and Bm3P1 (another small gene located upstream of the P450 (BM-3) gene that encodes a second putative regulatory protein) in resp onse to pentobarbital induction but left the basal levels unaffected, In gel mobility shift assays, Bm3R1, a repressor of the P450(BM-3), ge ne, was found to specifically interact with the Barbie box sequences o f the B, megaterium P450 genes, Mutated Barbie boxes showed a decrease d binding affinity for Bm3R1 compared to their wild type (unmutated) c ounterparts, Barbie box sequences were also shown to specifically inte ract with putative positive regulatory factors of B. megaterium cells, These putative positive factors were induced by pentobarbital and wer e also present at high levels during late stationary phase of B, megat erium cell cultures grown in the absence of barbiturates, The mutated Barbie box sequences had greater binding affinity for these positive f actors than did unmutated Barbie box sequences, DNase I footprinting a nalysis of the 5'-flanking region of the P450(BM-1) 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 footpr inting experiments with the 5'-flanking region of the P450(BM-3) gene failed, however, to unambiguously reveal protected sequences in the Ba rbie box region, The evidence suggests that the positive factors and B m3R1 compete with each other for binding to the Barbie box region, esp ecially in the 5'-flanking region of the P450(BM-1) gene, and for puta tive roles in the regulation of transcription from the B, megaterium P 450 genes, This inference was further supported by evidence derived fr om a nested-deletion analysis in and around the Barbie box of the P450 (BM-1) regulatory region that showed that a repressor binding site and a positive factor binding site overlap at the Barbie box sequence, In tote, these experimental results indicate that, in B. megaterium at l east, the Barbie box sequences are important cis acting elements for c oordinately regulating the barbiturate-mediated expression of the P450 (BM-1) and P450(BM-3) genes and the genes encoding their positive regu latory factors.