a, Cap dependent transcription assay (n=3) showing that 18 or 18+3 3′ vRNA templates are both active and produce the same transcription products. Read-through products and endonuclease cleavage products are indicated. Radiolabeled capped RNA markers, of size indicated, are at the left. An incubation time of four hours was chosen for the cryo-EM experiment as beyond that time there was no increase of product. b, Quantitative activity comparison of the 18 or 18+3 3′ vRNA templates after four hours in presence of ATP and GTP and 15-mer capped primer. The activity of 18+3 3′ vRNA is 72.8% ± 0.03 (n=6, mean and s.d.) of the activity of native 18 3′ vRNA. The read-through activity, defined by ratio of the read-through product to the desired capped transcription product, are 15% different for the two vRNA templates (n=6, mean and s.d., two-tail t-test). c, Cap dependent transcription assay showing products of 18 or 18+3 3′ vRNA templates with 14- and 15-mer cap primers. The capped 14-mer requires CTP in the reaction mixture to function as a primer, in accord with CTP serving as initiating nucleotide for this primer (although with the 18+3 template some mis-initiation seems to occurs, maybe because the primer and template anneal more readily).