| Description |
Studies in cells of all types indicate that DNA serves as the template for both its own replication as well as the synthesis of RNA. This observation, therefore, requires that the RNA-containing viruses must either coordinate or provide unique enzymology for the replication of their RNA genomes. One strategy which has developed to circumvent this dilemma has been the direct use of RNA as template for RNA synthesis. RNA-dependent RNA synthesis, while central to the genome replication of many important RNA viruses, remains largely biochemically uncharacterized. In an effort to elucidate the components and mechanism of RNA dependent RNA synthesis, an in vitro transcription system for polio RNA was developed and studied. The system consisted of the poliovirus RNA polymerase (3DPo1) and a "Host Factor (HF) fraction from uninfected cells. The transcription of polio RNA was strictly dependent on 3DPo1, HF, and a poliovirion RNA template. The transcription products contain both plus- (identical to the template) and minus- (complement of the template) strand RNA. The size of the product RNA was heterogeneous, the largest being apparently twice template length. The large product RNA results from 3DPol utilizing the 3' end of the template as a primer, therefore, generating product RNA covalently linked to the template. In an effort to study the mechanism involved in the production of template linked product RNA, the 3DPo1 and HF reactions were separated. Preincubation of template RNA with HF, followed by its removal by phenol extraction, results in a template modification which allows 3DPol to transcribe the RNA in the absence of added HF. Analysis of reinsulated as well as active HF mediated elongating template RNAi indicate the priming 3' ends are heterogeneous and are a result of random nucleolytic events. No evidence was found to imply that the natural 3' poly (A) end of the template was involved in self-priming. It is proposed that template cleavages adjacent to or in base paired regions of secondary structure, fortuitously provide template linked primers Pol for 3DPol, which when elongated appear to be up to dimer length.."" |
