Transcription of Double-Stranded RNA by Escherichia coli DNA-Dependent RNA Polymerase

Abstract

Double-stranded RNA of some virus genomes can be used as template for the DNA-dependent RNA polymerase purified from E. coli. The RNA synthesis requires all 4 nucleoside triphosphates and Mg2+ and is dependent on the presence of .sigma. subunit. The reaction is inhibited by rifampicin, streptolydigin and ethidium bromide, but not by DNase [EC 3.1.4.5] and actinomycin D which does not bind to double-stranded RNA. The template activity of double-stranded RNA from various viruses is different in each case. The order of template efficiency is Penicillium chrysogenum virus > cytoplasmic polyhedrosis virus > rice dwarf virus > reovirus. The product obtained using cytoplasmic polyhedrosis virus double-stranded RNA as template is single-stranded and hybridizes specifically to the denatured template RNA. One of the major 5''-starting nucleotide sequences of the product RNA is pppA-A-Y---. Transcription in vitro of double-stranded RNA by E. coli RNA polymerase is apparently initiated at specific sites on the template.