Fig. 3: smFRET studies of nascent riboSAM in ECs and isolated riboSAM without and with SAM at 2 mM Mg²⁺.

a 15-step reaction was performed to incorporate Cy3 (green star) and Cy5 (red star) into sites 37 and 74 for structural studies of nascent riboSAM in ECs. The ECs dissociated in steps 12, 13, and 14 were used for the co-transcriptional study, and the final product was used for the post-transcriptional study. The nucleotides transcribed in steps 12, 13, and 14 are shown in blue. SAM is shown as a red sphere. b Gel shift assays of EC-81, EC-86, EC-91 and the final product (37Cy3-74Cy5-riboSAM). The liquid phase separated by SPE at steps 12 to 15 was loaded at Lanes 1 to 4. The gels 1 and 2 were irradiated under 530 nm and 620 nm fluorescence, respectively. The gel 3 is the merged image of the gels 1 and 2. The experiment was repeated independently for three times. c–h smFRET histograms and transition density plots for EC-81 at 0 and 0.5 mM SAM (c and d), EC-86 at 0 and 0.5 mM SAM (e and f), EC-91 at 0 and 0.5 mM SAM (g and h). i, j smFRET histograms and transition density plots for the isolated 37Cy3-74Cy5-riboSAM at 0 and 0.5 mM SAM. Mean values ± s.d. of triplicate experiments are shown for peak percentages.