Extended Data Fig. 5: LC-MS analysis of IVT products with template gTT.
From: Molecular insight into 5′ RNA capping with NpnNs by bacterial RNA polymerase
The PAGE gel from Extended Data Fig. 4c is reproduced here for clarity. Extracted ion chromatograms of IVT products with and without addition of NpnNs. IVT products encoded from the +1 TSS are marked blue, IVT products encoded from the +1 TSS with an extra C added at the 3′ end are marked dashed blue. IVT products encoded from the −1 TSS are marked red, IVT products encoded from the −1 TSS with an extra C added at the 3′ end are marked dashed red. The sequences of the formed products with exact masses are shown in Supplementary Table 5. We confirmed the 16-mer product encoded from the −1 TSS and we detected a 17-mer RNA product corresponding with its mass also to the (−1)TSS-RNA but with an extra C added at the 3′ end. Only trace amounts of the 15-mer RNA encoded from the +1 TSS were detected by PAGE and LC-MS. The PAGE analysis of IVT reactions with Ap3-4A and the template gTT showed the formation of two capped products. LC-MS analysis confirmed the production of capped RNA encoded from the +1 TSS, but contrary to the control IVT, we did not detect capped RNA encoded from the −1 TSS. The second observed product has the mass of cap(+1)TSS-RNA with the 3′ extra C. These observations indicate that Ap3-4A initiate transcription only from the +1 TSS of the gTT template. The experiment with Ap3-4G led to the formation of similar products as in the case of Ap3-4A. As expected, Gp3-4G cannot base pair with the gTT template and did not produce capped RNA. N/D = not detected. Created in BioRender. Serianni, V. (2025) https://BioRender.com/jj7ixi3.
