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).

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.