Extended Data Fig. 6: In vitro effects of FluPol mutations involved in the interface to the Pol II EC.
From: Mechanism of co-transcriptional cap snatching by influenza polymerase
a-c, Effect of mutation of PA on the endonuclease activity of FluPol in vitro. a, Representative example for the denaturing urea PAGE of an endonuclease assay of FluPol interface1 mutants using the EC as a substrate. The substrate and the product bands are labeled. b, The fraction of cleaved RNA (intensity of cleaved product divided by intensity of all bands, see Extended Data Fig. 1c) is shown in dependence of the mutant. Each point reflects one experimental replicate (n = 5), shown are mean ± s.d. Significance p-values were calculated using a linear mixed-effects model (substrate as a fixed effect, experimental replicate as a random effect, two-sided, no multiple testing correction). Comparison of FluPol WT and FluPol PA Y131A or FluPol PA K104A, E141A have p-values of <0.0001 or 0.0062. c, Representative example for the denaturing urea PAGE of an endonuclease assay using the RNA only as a substrate shows similar activity of FluPol using ten times the amount of FluPol compared to the reactions in a. d-f, Effect of mutation of PB2 on the endonuclease activity of FluPol in vitro. d, Representative example for the denaturing urea PAGE of an endonuclease assay of FluPol interface 2 mutants using the EC as a substrate. The substrate and the product bands are labeled. e, The fraction of cleaved RNA (intensity of cleaved product divided by intensity of all bands, see Extended Data Fig. 1c) is shown in dependence of the mutant. Each point reflects one experimental replicate (n = 4), shown are mean ± s.d. f, Representative example for the denaturing urea PAGE of an endonuclease assay using the RNA only as a substrate shows similar activity of FluPol using ten times the amount of FluPol compared to the reactions in d.
