Phenobarbital-dependent protein binding to Barbie box-like sequences in the coding region of cytochrome P450 BM-3 gene from Bacillus megaterium

Abstract

Phenobarbital-dependent protein binding was shown to occur to DNA fragments from the coding region of the cytochrome P450 BM-3 gene from Bacillus megaterium. Incubation of the DNA fragments from the coding region of the gene with total cell extract from Bacillus megaterium revealed two DNA regions with protein-binding capacity: +237 +318 and +319 +425 considering ‘0’ as the start of cytochrome P450 BM-3 translation. DNaseI footprint analysis of the fragment +319 +425 with the total cell extract showed that some protein(s) protected DNA stretches from the position +373 up to the position +389 on the transcribed strand and from the position +378 up to the position +398 on the non-transcribed strand. DNaseI footprint analysis of the fragment +237 +318 revealed the protection in the region +262 +277 on the non-transcribed strand. Three regions protected by cell extract protein(s) from DNaseI hydrolysis ( +262 +277 , +373 +389 and +378 +398 ) appeared to be strongly homologous to the Barbie box sequence. Barbie-box-like sequences were found in the majority of regulatory regions of phenobarbital-inducible genes whose regulatory sequences had been reported (Fulco et al., 1994). Our results suggest that a functional role of Barbie box sequence takes place not only in regulatory but also in the coding region of the gene. In line with that hypothesis we analyzed all cytochrome P450 genes in respect to the presence of Barbie box-like sequences in their coding parts. At least one cytochrome P450 gene ( CYP6A1, phenobarbital-inducible gene from Musca domestica) was shown to contain Barbie box sequence in the coding part of the gene.