Fig. 2.

Schematic representation of a new method for the analysis of the premature transcription termination transcriptional activity and primers/oligomer design. mRNA (violet/red line) is cleaved by RNase H (pink circle) downstream the riboswitch (red line), in a position indicated by a DNA oligomer. Transcript levels are measured in a ddPCR reaction, preceded with the reverse transcription. First pair of primers (F1/R1) is complementary to the region upstream of the riboswitch sequence (5′ UTR and riboswitch), and therefore, it allows for the amplification of the full-length and terminated transcripts. The second pair of primers (F2/R2) flanks the RNase H cleavage site and serves as a control of the RNase H cleavage efficiency. The third pair of primers (F3/R3) is complementary to the region downstream the riboswitch sequence (ORF and 3′ UTR), and it allows for amplification of the full-length transcripts solely