Biomaterial Database

Inhibition by barbiturates of the binding of Bm3R1 repressor to its operator site on the barbiturate-inducible cytochrome P450BM-3 gene of Bacillus megaterium. 1993

G C Shaw, and A J Fulco

Department of Biological Chemistry, UCLA School of Medicine 90024-1737.

In our previous publication (Shaw, G.-C., and Fulco, A. J. (1992) J. Biol. Chem. 267, 5515-5526), we reported that Bm3R1, a protein encoded in an open reading frame just upstream from the cytochrome P450BM-3 gene, is a repressor critically involved in the barbiturate-inducible expression of this gene in Bacillus megaterium. We now describe the purification of the Bm3R1 protein from an overproducing Escherichia coli strain harboring a bm3R1 gene-carrying plasmid and report the effect of barbiturate inducers on the interaction of Bm3R1 with a fragment of B. megaterium DNA containing the bicistronic operator and promoter sequences. Gel filtration analysis revealed that, under our experimental conditions, most of the Bm3R1 protein exists in highly aggregated forms. Gel mobility shift assays showed that Bm3R1 protein bound specifically to a segment of DNA containing the promoter-operator region of the bm3R1 gene while DNase I footprinting experiments established that Bm3R1 protected a region of DNA that covers and flanks the palindromic operator sequence. The interaction between Bm3R1 repressor and its operator, in vitro, was strongly inhibited by the addition of 2 mM pentobarbital or 2 mM methohexital (strong in vivo inducers of P450BM-3) but not by the same concentration of phenobarbital (a relatively weak inducer) or by mephobarbital (a non-inducer). A detailed comparison of pentobarbital and methohexital at concentrations lower than 2 mM indicated that methohexital was 5-10 times more effective as an inhibitor of Bm3R1 binding in vitro, as compared with its 7-fold greater inducer potency in vivo. The observation that the in vitro inhibition effects of barbiturates on the interaction of Bm3R1 repressor and its operator correlate strongly with their in vivo potency as inducers of cytochrome P450BM-3 suggests a mechanism for the induction process. It seems plausible that the barbiturate inducers might bear a conformational resemblance to and mimic the mode of action of an as yet unidentified endogenous inducer(s) in B. megaterium that functions by releasing the binding of Bm3R1 repressor from its operator site.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009875	Operator Regions, Genetic	The regulatory elements of an OPERON to which activators or repressors bind thereby effecting the transcription of GENES in the operon.	Operator Region,Operator Regions,Operator, Genetic,Genetic Operator,Genetic Operator Region,Genetic Operator Regions,Genetic Operators,Operator Region, Genetic,Operators, Genetic,Region, Genetic Operator,Region, Operator,Regions, Genetic Operator,Regions, Operator
D010424	Pentobarbital	A short-acting barbiturate that is effective as a sedative and hypnotic (but not as an anti-anxiety) agent and is usually given orally. It is prescribed more frequently for sleep induction than for sedation but, like similar agents, may lose its effectiveness by the second week of continued administration. (From AMA Drug Evaluations Annual, 1994, p236)	Mebubarbital,Mebumal,Diabutal,Etaminal,Ethaminal,Nembutal,Pentobarbital Sodium,Pentobarbital, Monosodium Salt,Pentobarbitone,Sagatal,Monosodium Salt Pentobarbital
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D012097	Repressor Proteins	Proteins which maintain the transcriptional quiescence of specific GENES or OPERONS. Classical repressor proteins are DNA-binding proteins that are normally bound to the OPERATOR REGION of an operon, or the ENHANCER SEQUENCES of a gene until a signal occurs that causes their release.	Repressor Molecules,Transcriptional Silencing Factors,Proteins, Repressor,Silencing Factors, Transcriptional
D003577	Cytochrome P-450 Enzyme System	A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism.	Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D004268	DNA-Binding Proteins	Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.	DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
D005798	Genes, Bacterial	The functional hereditary units of BACTERIA.	Bacterial Gene,Bacterial Genes,Gene, Bacterial
D001410	Bacillus megaterium	A species of bacteria whose spores vary from round to elongate. It is a common soil saprophyte.	Bacillus megatherium
D001426	Bacterial Proteins	Proteins found in any species of bacterium.	Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial