Extended Data Fig. 6: IVT with NpnNs and template CT.
From: Molecular insight into 5′ RNA capping with NpnNs by bacterial RNA polymerase
a, Schemes of IVT experiments showing NTPs or NpnNs binding to the template CT (with a pre-melted bubble from position −11 to +2) during transcription initiation with different base pairing at the −1 and +1 positions. b, Sequences of color-coded RNA products with and without the addition of NpnNs. Uncapped 15-mer referred to as (+1)TSS-RNA is indicated by a blue dot, uncapped 16-mer referred to as (−1)TSS-RNA is indicated by a brown dot, capped 15-mer referred to as cap(+1)TSS-RNA is indicated by a green dot, and capped 16-mers referred to as cap(−1)TSS-RNA are indicated by red and yellow dots. c, PAGE (with APB) showing RNA products from IVT using the template CT with NTPs or various NpnNs, radiolabeled with α-[32P]CTP. Each control (CTRL AG, CTRL GG, CTRL AA, Supplementary Table 2) shows the migration of the uncapped RNA 15-mer and 16-mer and serves as size marker (panel d). IVT reaction (Extended Data Table 1) without NpnNs led to the production of mainly 16-mer and also 15-mer uncapped products. The RNA products are marked according to panel b. We observed formation of capped 15-mer RNA encoded from the +1 T position when Ap3-4A were added to IVT. When Ap3-4G and Gp3-4G were added, we observed mainly capped 16-mer RNA encoded from the −1 C position of the template. This indicates that the transcription was initiated by the G nucleoside of GpnNs bound at the −1 C position of the template. The IVT was performed in ten independent experiments; one representative gel is shown. d, PAGE (without APB) shows migration of 15-mer (p-MARKER(15)) and 16-mer (p-MARKER(16)) monophosphate marker along with the monophosphate RNA (p-CTRL) and triphosphate RNA (ppp-CTRL) generated using template CT and regular NTPs. The experiment was performed in triplicate. Created in BioRender. Serianni, V. (2025) https://BioRender.com/opxr6fh.
