Sequence and analysis of the gene for bacteriophage T3 RNA polymerase

Abstract

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.

Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polyamine biosynthesis in Escherichia coli

“…Spoerel and Herrlich (43) reported the copurification of two T3 AdoMetase proteins differing in molecular weight by roughly 3 kDa, while the T7 gene encodes and produces a single peptide. These observations lead us to suspect that unlike the highly similar T3 and T7 phage RNA polymerases (28) and other regions of the phage genomes (12), the two anti-restriction proteins are quite different. These conclusions are supported by preliminary data from DNA sequencing studies and may help us to better appreciate the evolutionary and structural relationships between the two phages, the mechanism behind their genetic divergence, and the nature of viral evolution in general.…”

DNA Sequence Analysis of Regions SurroundingblaCMY-2from MultipleSalmonellaPlasmid Backbones

“…The genomic library was transformed into E. coli Top10 cells (Invitrogen), and random clones (approximately 4ϫ coverage) were sequenced on a LiCor (Lincoln, Nebr.) 4000 sequencer by using IR800-labeled T3 and T7 primers (13,21). Contigs were generated by using Vector NTI Contig Express software (Informax, North Bethesda, Md.…”

Promoter specificity determinants of T7 RNA polymerase

“…18 and D. Parrotta, personal communication) were digested with EcoRV and SspI, treated with proteinase K, extracted with phenol and chloroform, and precipitated with ethanol (26). Transcription reactions were carried out in a volume of 10 l in 30 mM Hepes, pH 7.8, 100 mM potassium glutamate, 15 M Mg(OAc) 2 , 0.25 mM EDTA, 1 mM DTT, 0.05% Tween-20 (27) containing 0.5 mM ATP, CTP, GTP, and UTP (Pharmacia Ultrapure), 2 Ci of [␣- 32 P]ATP (specific activity 800 Ci͞mmol; New England Nuclear), 10 ng of RNA polymerase, and 0.3 g of each plasmid template (for mixed template reactions) or 0.5 g of a single plasmid (for reactions having an individual template). Reactions were incubated at 37°C for 10 min, and the products were analyzed by electrophoresis in polyacrylamide gels containing 7 M urea as previously described (25).…”