Figure 5.
Swapping of the genomic and antigenomic hairpin structures at the transcriptional start site. (A) Scheme of hairpin swapping (nucleotides 56–78; see also Fig. 3 and 4) between the genomic and antigenomic substrates. The parental substrates were (−) RNA 154–55 and (+) RNA P_56–158 and the hybrid substrates were termed RNA 154–55(+hp) and RNA (-hp)56–158. Note that in the chimeric RNAs the (−) RNA hairpin was transplanted from the 3′- to the 5′-end and the (+) RNA hairpin vice versa. (B) EMSA-based VP30 binding curves and derived appKd values (± SD) for the RNA substrates illustrated in panel A as well as additional control RNAs for comparison. Each curve is based on at least 3 independent experiments (error bars are standard deviations of the mean); curves were fitted according to a one-ligand-binding-site model. Note that RNA 80–46 had 5 additional single-stranded nucleotides at the 3′-end owing to linearization of the T7 transcription template with EcoR1 (see Supplementary material).