Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters

The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions. To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal. The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined. The results indicate that specific contacts within the major groove of the promoter between positions-5 and -12 are important for phage polymerase binding. Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerase-promoter complexes.     

SP6 RNA polymerase efficiently synthesizes RNA from short double-stranded DNA templates.

SP6 DNA-dependent RNA polymerase, like T7 RNA polymerase, can be used to synthesize RNA sequences from short DNA templates which contain the 18 base pair promoter region. Use of SP6 polymerase extends the range of possible 5' sequences of RNA products, since the preferred SP6 start site (of the RNA product) is 5'GAAGA, while T7 polymerase prefers 5'GGGAG. The SP6 start site can be advantageous in large-scale syntheses where high concentrations of RNA can lead to aggregation. Using the limited number of DNA templates described here, there appears to be a significant difference between the two enzymes: SP6 polymerase requires a complete duplex DNA substrate for efficient synthesis, unlike the T7 enzyme which works efficiently when only the 18 base promoter region is double-stranded. SP6 polymerase consistently produces higher yields of RNA than does T7 polymerase, and the reactions can be easily scaled up to produce milligram quantities of RNA.     

Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity.

The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.     

Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a ''stretched'' preproparathyroid hormone.

The intergenic region of bacteriophage f1 has been subcloned into the bacteriophage SP6 promoter plasmids, pSP64 and pSP65, in both orientations. Coinfection of E. coli with these SP6 promoter/phage f1 chimeric plasmids and the interference resistance phage, IR1, results in the replication and secretion of the pSP6.f1 plasmids as single stranded DNA. Bovine preProPTH cDNAs in both the native form and a form containing an insertion of 117 base pairs in the protein coding region have been inserted in these plasmids. The RNA transcribed from the SP6.f1/preProPTH cDNA constructs was efficiently translated in the wheat germ or reticulocyte cell free systems without addition of a 7-methylguanosine cap to the RNA. In the presence of dog pancreatic or chicken oviduct microsomal membranes, conversion of the resultant pre-proteins to pro-proteins was observed. Confirmation of the "mutated" preProPTH cDNA was determined by dideoxyribonucleotide DNA sequencing of single stranded plasmid DNA. These vectors are suitable for the efficient biosynthesis of large amounts of single or double stranded DNA, and translationally active RNA. The combined properties of single stranded DNA replication and the SP6 promoter simplify the engineering of mutant RNAs and their corresponding proteins. In addition, single stranded DNA or RNA corresponding to either complementary strand may be synthesized as nucleic acid hybridization probes.