Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence

Abstract

We describe an in vitro transcription system in which the polyadenylated 3' ends of vaccinia virus mRNAs are formed by termination downstream of a regulatory signal. When linear DNA templates containing vaccinia early genes were incubated with soluble enzymes extracted from vaccinia virions, mature-size mRNAs were synthesized within 1 min, whereas longer run-off transcripts were not detected until later. Deleting a series of thymidylate residues located downstream of the coding region abolished termination. The 3' ends of the transcripts formed heterogeneously, between 50 and 70 nucleotides past the T-rich termination signal, in either viral- or vector-derived sequences. Polyadenylation of the transcripts occurred without regard to the terminal sequence and was inhibited by adding exogenous RNA. Although exogenous RNAs were also polyadenylated, processing of 3' ends was not observed.