Characterization of T7-specific Ribonucleic Acid Polymerase

Abstract

The biochemical properties and template specificity of the T7 phage-specific DNA-dependent RNA polymerase have been studied. The enzyme shows the same absolute requirements for DNA, nucleoside triphosphates, and a divalent metal ion as are found for bacterial RNA polymerases, however, the conditions for optimal RNA synthesis are somewhat different. The T7 RNA polymerase initiates T7 RNA chains rapidly with GTP; each enzyme molecule can initiate several T7 RNA chains in the course of the reaction. Hence, an efficient termination mechanism is present in the in vitro system. Of the helical DNAs tested, only T7 DNA, T3 DNA, and salmon sperm DNA have appreciable template activity with T7 RNA polymerase. A variety of single-stranded and denatured DNAs support RNA synthesis but at a reduced rate. Of the helical synthetic polynucleotides tested only d(I)n·d(C)n and d(G)n·d(C)n were active; both support the synthesis of poly[r(G)]. In contrast, both single-stranded poly[d(C)] and poly[d(T)] served as templates. It is suggested that T7 RNA polymerase requires a specific promoter site on DNA for effective transcription; this site is different from that used by bacterial RNA polymerase and may be rich in cytosine and thymidine residues.