Coupling of replication to transcription in vitro

Abstract

In a coupled system consisting of RNA polymerase and DNA polymerase I of Escherichia coli, the four deoxyribo- and the four ribonucleoside triphosphates, and DNA of bacteriophage f1 as template, DNA synthesis depends on the concomitant synthesis of RNA. Over a wide range of concentrations of the two polymerases, RNA synthesis was unaffected by the simultaneous synthesis of DNA, whereas the rate of DNA synthesis depended on the level of RNA synthesis. In the coupled reaction, RNA synthesis starts immediately at a high rate, which subsequently decreases, whereas DNA synthesis starts after a lag and its rate increases as the reaction proceeds. Upon addition of rifampicin, the rate of RNA synthesis falls abruptly, while that of DNA declines only gradually. The base composition of the DNA synthesized in the coupled reaction is complementary to that of f1 DNA template. It is suggested that the RNA synthesized by the RNA polymerase serves as a primer rather than as a template for the DNA polymerase.