Fidelity of in Vitro Transcription of T3 Deoxyribonucleic Acid by Bacteriophage T3-induced Ribonucleic Acid Polymerase and by Escherichia coli Ribonucleic Acid Polymerase

Abstract

Abstract The fidelity of in vitro transcription of bacteriophage T3 DNA by T3 RNA polymerase and Escherichia coli RNA polymerase have been studied by DNA-RNA hybridization-competition studies with regard to (a) asymmetry of transcription and (b) the region of the T3 DNA transcribed by either polymerase. RNA transcribed by T3 RNA polymerase hybridized exclusively with the H strand of T3 DNA—the only strand that is copied in vivo at all times following T3 phage infection. In contrast, RNA transcribed by E. coli RNA polymerase hybridized with both the H and the L strands of T3 DNA, although a high proportion of RNA chains (g70%) hybridized with the H strand. Approximately 15 to 20% of RNA chains hybridized with the L strand, the strand that does not appear to be copied in vivo. The ratio of H to L strand copying by E. coli polymerase was unaltered by the presence of either excess σ or ρ factor or by changes in the ratio of polymerase to DNA in the reaction mixture. Competition-hybridization studies between RNA synthesized in vitro and RNA isolated after T3 phage infection indicate that RNA transcribed in vitro by T3 RNA polymerase contained all of the sequences present in late in vivo RNA. In addition, all sequences present in early in vivo RNA were also present in in vitro T3 RNA polymerase products. Thus, in vitro, T3 RNA polymerase transcribed the entire early and late regions of the T3 genome. RNA transcribed by E. coli RNA polymerase contained all sequences present in early in vivo RNA. In addition, such in vitro RNA also contained nearly 50% of the sequences present in late in vivo RNA. In contrast, RNA transcribed by E. coli RNA polymerase in the presence of termination factor ρ contained all of the sequences present in early in vivo RNA, but contained very few late RNA sequences. These results demonstrate that E. coli RNA polymerase, in the absence of ρ factor, can read through early termination signals to transcribe part of the late regions. The presence of ρ factor restricts E. coli RNA polymerase to copying primarily early regions. In agreement with the above results, it was observed that RNA synthesized either by T3 RNA polymerase or by E. coli RNA polymerase could direct in vitro synthesis of both early and late T3 phage-specific enzymes—S-adenosylmethionine-cleaving enzyme and lysozyme, respectively—in an in vitro protein-synthesizing system. When RNA transcribed by E. coli RNA polymerase in the presence of ρ factor was used as messenger, lysozyme synthesis was markedly depressed.