RNA Polymerase Binding Sites of Phage fd Replicative Form DNA

Abstract

WHEN RNA polymerase binds to DNA templates, a fraction of DNA is protected from nucleolytic digestion. This fraction has been assumed to represent the binding sites of RNA polymerase^1–6. There is, however, no direct evidence that the isolated fragments really contain specific sites at which transcription is correctly initiated. An approach to this question is to demonstrate that the specificity of transcription is still retained in the isolated fragments. RNA chains transcribed in vitro on the doubly closed replicative form DNA (RF-I DNA) of bacteriophage fd are predominantly initiated with pppAUG—, pppGUA— and pppGUU—⁷ Accordingly, the DNA fraction protected by RNA polymerase against the nucleolytic digestion was isolated from the RF-I DNA RNA polymerase complex, and the template activity of the isolated fragments was examined.