Transcription of Double-Stranded Viral and Cellular DNAs by Purified Mammalian DNA-Dependent RNA Polymerases

Abstract

The multiplicity and different intranuclear localizations of DNA-dependent RNA polymerases have suggested that gene expression in animal cells is regulated, at least in part, by distinct RNA polymerases with different template specificities (1). One of the most direct ways to support this hypothesis would be to show in vitro that the various purified enzymes specifically transcribe different parts of the deproteinized chromosomal DNA. A first step in such a study is to ask whether the purified enzymes alone can in fact initiate RNA synthesis on an intact double-stranded DNA or whether the presence of additional initiation factor(s), similar to the Escherichia coli o factor (2,3), is required. Previous studies involving normal calf thymus DNA preparations and the initiation inhibitor AF/013 (4-7) have suggested that purified AI and B enzymes initiate at different sites on calf thymus DNA and that the B enzymes could in fact initiate on “true” double-stranded regions of the DNA, while AI enzyme could lack an initiation factor. It is nevertheless clear that the possible specificity of the animal enzymes cannot be unequivocally demonstrated using these ordinary preparations of DNAs, since all of them contain a high number of single-stranded nicks (“high” compared to the expected number of true promoter sites), acting as efficient nonspecific initiation sites (8,9).