Transcriptional regulation of the genes encoding cytochromes P450BM-1 and P450BM-3 in Bacillus megaterium by the binding of Bm3R1 repressor to Barbie box elements and operator sites.
We previously reported (Liang, Q., He, J.-S., and Fulco, A.J. (1995) J. Biol. Chem. 270, 4438-4450) that Bm3R1, a repressor regulating the expression of P450BM-3 in Bacillus megaterium, could bind to Barbie box sequences in the 5'-flanking regions of barbiturate-inducible genes. We've now shown that pentobarbital does not inhibit in vitro binding of Bm3R1 to the P450BM-3 and P450BM-1 Barbie boxes (BB3 and BB1), although the palindromic operator sequence (OIII) of P450BM-3 did have a strong competitive effect on such binding. G39E-Bm3R1, a mutant of Bm3R1, did not bind to either Barbie box. In the presence of Bm3R1, portions of the regulatory regions of P450BM-3 and P450BM-1 were protected from DNase I digestion. These included 11 of the 15 base pairs of BB3 plus 7 base pairs 3' to BB3, BB1 plus 16 base pairs 3' to BB1, and, in the 5'-flanking region of P450BM-1, segments covering most of two palindromic sequences (OII and OIII) of 24 and 52 base pairs. These DNase I-protected regions (including OIII) showed considerable sequence identity, especially in a conserved poly(A) motif. Barbiturates did not inhibit binding of Bm3R1 to OI. OII in vitro while G39E-Bm3R1 did not bind. The regulatory effects of Bm3R1 on P450BM-1 and P450BM-3 were also evaluated in vivo using heterologous chloramphenicol acetyltransferase constructs and Western blotting. In the G39E mutant strain, both P450BM-1 and P450BM-3 were constitutively expressed, and the regulatory proteins Bm1P1 and Bm3P1, although still pentobarbital-inducible, had significantly higher basal levels of synthesis. In toto, our results show that Bm3R1 represses both P450BM-1 and P450BM-3 expression and that it may effect this by coordinate binding to operator and Barbie box sequences to produce looping of the P450BM-1 and P450BM-3 regulatory regions through protein-protein interaction.
Liang
Q
Q
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1737, USA.
Fulco
A J
AJ
D001410:Bacillus megaterium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism*; D001463:Barbiturates / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001463:Barbiturates; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C073493:Bm3R1 protein, Bacillus megaterium; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases