Molecular characterization of an RNA-dependent transcription reaction catalyzed by RNA polymerase II

The specificity of the initiation event of RNA transcription from DNA templates resides in the specificity of interaction of the machinery with DNA promoter elements. In the case of eukaryotic nuclear RNA polymerase II (PolII), the complex machinery responsible for accurate initiation and elongation of transcription is composed of approximately 30 polypeptides. Still, the first and rate-limiting step in the transcription event from most PolII-transcribed genes studied is the specific interaction of a component of this machinery with the cognate DNA sequence at the promoter region.;The question arises whether RNA molecules of particular sequence or structure could usurp DNA-templated RNA polymerases to transcribe complementary copies of themselves. A priori, the remarkable structural possibilities open to RNA would sustain such scenario. Indeed, a class of RNA pathogens of plants (viroids) and their possible counterpart in animals (Hepatitis Delta Virus: HDV) depend on host activities for the replication of their RNA genome into RNA copies. PolII has been suggested as the activity responsible for their replication in plant and animal cells, respectively.;I have chosen HDV as model system to study how an RNA molecule can redirect a DNA-dependent RNA polymerase to specifically initiate transcription from RNA templates. For this, I have exploited a transcription system in vitro in which segments of HDV RNA can serve as templates for PolII transcription. By the reductionist methodology of biochemical fractionation/reconstitution, I have investigated which factors are necessary and sufficient to support such specific transcription in vitro. I have arrived at a minimal system of highly purified core PolII and a recombinant preparation of the cofactor PC4 which recapitulates most of the characteristics of the reaction carried out in nuclear extracts.;The characterization of the template requirements, the nature of the product of such reaction, and the components of the machinery has allowed me to generate a number of models not only concerning possible aspects of regulation of the replicative cycle of this virus, but also on some possible novel mechanisms of control of canonical DNA-templated transcription by PolII mediated by the cofactor PC4.