Fig. 1: IVT reactions with NpnNs and template TC.
From: Molecular insight into 5′ RNA capping with NpnNs by bacterial RNA polymerase
a, Chemical structure of NpnNs. b, Schemes of IVT experiments where NTPs or NpnNs bind to the template TC (with a premelted bubble from position −11 to +2) during transcription initiation with different base-pairing combinations between NpnNs and the −1 and +1 template strand positions. T, template; NT, nontemplate. c, 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-mers referred to as cap(+1)TSS-RNA are indicated by green and yellow dots and capped 16-mers referred to as cap(−1)TSS-RNA are indicated by purple and pink dots. d, PAGE analysis with acryloylaminophenyl boronic acid (APB) showing RNA products obtained from IVT reactions with the template TC and NTPs or their combination with various NpnNs radiolabeled with α-[32P]CTP. Each control (CTRL AG, CTRL GG, CTRL AA) was designed according to Supplementary Table 2; it shows the migration of the uncapped RNA 15-mer (blue dots) and 16-mer (brown dots) and serves as a molecular weight marker (e). The capped products are marked with colored dots according to c. The IVT was performed in ten independent experiments; one representative gel is shown. Nonadjacent lanes from the same experiment were rearranged and are separated by delineating black lines. Each lane originates from the same gel. e, PAGE (without APB) analysis showing the migration patterns of a 16-mer and 15-mer monophosphate marker along with the triphosphate RNA (ppp-CTRL) and the monophosphate RNA (p-CTRL) generated using template TC and regular NTPs. The markers have the same sequence as the RNA transcript expected from the template TC (Extended Data Table 1), starting at position −1 (p-MARKER(16)) and +1 (p-MARKER(15)). This experiment was performed in triplicate; one representative gel is shown. Created in BioRender. Serianni, V. (2025) https://BioRender.com/p388ku2.
