Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity.

Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters. A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches. First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping. This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623. To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constructed by in vitro manipulation of the cloned genes. The specificity of the resulting hybrid RNA polymerases in vitro and in vivo indicates that an interval of the polymerase that spans amino acids 674 to 752 (the 674 to 752 interval) contains the primary determinant of promoter preference. Within this interval, the amino acid sequences of the T3 and T7 enzymes differ at only 11 out of 79 positions. It has been shown elsewhere that specific recognition of T3 and T7 promoters depends largely upon base-pairs in the region from -10 to -12. An analysis of the preference of the hybrid RNA polymerases for synthetic T7 promoter mutants indicates that the 674 to 752 interval is involved in identifying this region of the promoter, and suggests that another domain of the polymerase (which has not yet been identified) may be involved in identifying other positions where the two consensus promoter sequences differ (most notably at position -15).

Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate.

Initiation of RNA synthesis by the phage polymerases is abortive if the concentration of pyrimidine triphosphates is limiting. Under abortive initiation conditions the polymerases repeatedly initiate transcription but produce ribooligonucleotides that terminate just prior to the first occurrence of the limiting substrate. Abortive initiation is most severe if the limiting substrate occurs within the first 8-12 nucleotides of the nascent RNA chain and is particularly evident when UMP is limiting. The formation of stable elongation complexes (as determined by gel retardation experiments) occurs after the synthesis of an RNA product 8-12 nucleotides in length.

Sequence and analysis of the gene for bacteriophage T3 RNA polymerase.

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.

Studies on chiral interactions of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane and the corresponding N-hydroxy metabolites with cytochrome P-450.

The stereoselective pharmacological behavior and metabolism of the potent psychotomimetic amine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane have led to an investigation of the interactions of the racemic amine, its enantiomers, and the corresponding N-hydroxy metabolites with rabbit liver microsomal cytochrome P-450. An examination of the formation of cytochrome P-450 metabolic intermediate complexes with these species suggests that N-oxidation of the pharmacologically active (R)-amine in inhibited by the S enantiomer. Additionally, metabolic intermediate complex formation [favored by the (R)-amine] appears to be associated with loss of microsomal mixed function N-oxidase activity. The results have led to the prediction that N-hydroxylation of pure (R)-amine may be a qualitatively more important pathway than that observed with racemic amine even though this biotransformation may be suicidal.

In vitro stereoselective metabolism of the psychotomimetic amine, 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane. An apparent enantiomeric interaction.

The stereoselective metabolism [R/S (metabolized) less than 1] of the psychotomimetic amine (R,S)-1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane in 10 000g rabbit liver homogenate supernatant and 100 000g microsomal fractions has been demonstrated with the aid of the chiral reagent (S)-N-pentafluorobenzoylprolyl-1-imidazolide and GLC analyses. In contrast to the enantiomeric discrimination observed with racemic amine, the individual isomers were metabolized at approximately the same rate. This apparent enantiomeric interaction illustrates the fact that racemates should be viewed as unique chemical species with pharmacodynamic and toxicologic profiles potentially different from the individual antipodes.