The molecular cloning and characterization of BM1P1 and BM1P2 proteins, putative positive transcription factors involved in barbiturate-mediated induction of the genes encoding cytochrome P450BM-1 of Bacillus megaterium

Abstract

Analysis of a 1.3-kilobase segment of 5'-flanking DNA from the barbiturate-inducible P450BM-1 gene (CYP106) of Bacillus megaterium revealed two open reading frames. One, BM1P1, encodes 98 amino acids and is located 267 base pairs upstream from the sequence encoding cytochrome P450BM-1 but in the opposite orientation. The second, BM1P2 (88 amino acids), is 892 base pairs upstream from the P450BM-1 coding sequence and in the same coding strand. The expression of BM1P1 and BM1P2 was strongly stimulated in cells grown in the presence of pentobarbital, and the BM1P1 gene product exerted positive control on expression of P450BM-1. When a 177-base pair fragment encompassing the overlapping promoter regions of the P450BM-1 and BM1P1 genes was used as a probe in DNA binding assays, the BM1P1 and BM1P2 gene products and Bm3R1 (the repressor protein regulating the barbiturate-mediated expression of P450BM-3) could bind individually, but the addition of BM1P1 or BM1P2 to a binding mixture containing Bm3R1 completely prevented the appearance of a Bm3R1 binding band. When a 208-base pair fragment containing a Barbie box sequence and located upstream of the 177-base pair fragment was used as a probe, only a Bm3R1 binding band was detected. Although neither BM1P1 and BM1P2 appeared to bind to this 208-base pair fragment, their presence strongly inhibited the binding of Bm3R1 to the same probe. The evidence suggests that BM1P1 and BM1P2 may, in part, act as positive regulatory proteins involved in the expression of the P450BM-1 gene by interfering with the binding of the repressor protein, Bm3R1, to the regulatory regions of P450BM-1.