Fig. 4. PQS orientation mediates elongation events by dual-PIFE assay.

a Experimental scheme of dual-PIFE assay. The two dyes are separated out of FRET-sensitive distance. Dual excitation of 532 nm (green) and 641 nm (red) lasers are applied simultaneously. b Trace examples demonstrate the events of single PIFE (green only) and dual-PIFE (green and red). c Examples of both single- (green) and dual-PIFE signal (red) that can exist in the same trace, indicating elongation is not always successful. d The fraction of successful elongation event is calculated by counting the number of dual-PIFE events out of total events. Data are presented as mean ± SEM of n = 7 independent experiments. Statistical significance, ***P < 0.001 was performed by one-sided Pearson’s χ²-test. Exact mean and P-value are provided in Supplementary Table 3.7.