Figure 4

Detection of RNA structure rearrangement during SMRT reverse transcription. (a) Histogram of reverse transcript lengths on E. coli 16S rRNA. The top axis shows 16S rRNA sequence position as defined by EcoCyc [17]. The left inset shows a schematic of 16S rRNA secondary structure. A section of 16S rRNA (gray rectangle) is expanded in the right inset, showing the start of SMRT reverse transcription and the 5’-end of 16S rRNA. Reverse transcribed bases are shown as solid blue circles and bases involved in secondary structures in transparent blue circles. Peaks in the histogram and their corresponding 16S rRNA sequence positions are indicated by arrows. (b) Histogram of reverse transcript lengths on human ribosomal protein S17 mRNA. Inset: Pausing probabilities along the mRNA template (blue bars), compared to calculated base-pairing probabilities of this mRNA template using models that do not allow (red) or do allow (black) for refolding of the RNA template remaining at each position in the DNA synthesis (Kinetic Trap Model and Equilibrium Model, respectively). A pause along mRNA template during SMRT reverse transcription was defined as stalling of reverse transcription for longer than 5 min (Methods).