Transcription of denatured T 4 DNA

Abstract

The synthesis of RNA is described, using denatured T 4 DNA and Micrococcus lysodeikticus RNA polymerase. In the reaction mixture resistance of newly synthesised RNA to ribonuclease is initially very high (70–80 %) and falls with time of incubation to reach a steady level in the region of 50%. A DNA-RNA duplex is synthesised, its RNA/DNA ratio rasing to a maximum of about 0.5. Single-stranded RNA and RNA-RNA duplex are also synthesised. Both DNA-RNA and RNA-RNA duplexes show reasonably sharp melting profiles. All molecules of DNA are involved in duplexes with RNA. When the DNA is broken to 6-S fragments, no separation of DNA from DNA-RNA hybrid is observed, showing that each DNA fragment carries RNA. It is proposed that approximately half the DNA between adjacent polymerase attachment sites is transcribed. Measurements by DNA-RNA hybridisation of the homology between messenger RNA and RNA synthesised on the denatured template are in agreement with this. When preformed DNA-RNA hybrid is reincubated with RNA polymerase, RNA is released from the hybrid by a process which is dependent upon the occurrence of RNA synthesis. The RNA released is partly ribonuclease sensitive, partly resistant. On the basis of these experiments a mechanism is proposed to explain the synthesis of RNA-RNA duplex.