Translational mapping of bacteriophage T7 RNAs synthesized In vitro by purified T7 RNA polymerase

Late bacteriophage T7 RNA, synthesized in vitro from wild-type, amber and deletion mutant phage DNAs using purified T7 RNA polymerase, was used to program an RNA-dependent in vitro protein synthesizing system. Products were analyzed by electrophoresis on polyacrylamide gels. When unfractionated late T7 RNA was translated, almost all late T7 proteins were synthesized, demonstrating that the entire late region of T7 DNA is transcribed by T7 RNA polymerase in vitro and that the leftmost initiation site for T7 RNA polymerase lies between the left ends of genes 1 and 1.3. Translation of fractionated species of in vitro synthesized late T7 RNA, purified by polyacrylamide gel electrophoresis, showed that T7 RNA species IV and V stimulate the synthesis of the T7 genes 9 and 10 proteins and of the gene 10 protein, respectively. Species VI does not encode any T7 proteins. The major products of translation of species III are genes 9, 10 and 17 polypeptides. These results demonstrate the existence of promoters for T7 RNA polymerase between genes 8 and 9 and between genes 9 and 10, and a termination site between genes 10 and 11. They are also consistent with the finding (Golomb & Chamberlin, 1974b) that RNA species IIIb and VI are transcribed from the extreme right end of T7 DNA.