Extended Data Fig. 8: Analysis of the role of the Trl1-LIG C-terminal domain during RNA ligation.
From: Structure of fungal tRNA ligase Trl1 with RNA reveals conserved substrate-binding principles
a, Active site adenylylation of WT CtTrl1-LIG and the ΔCTD variant was monitored by incorporation of αP32-labelled ATP. The time course of the adenylylation reaction was analyzed by autoradiogram (upper panel). Even protein loading was confirmed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (lower panel). b, Quantification of the experiment in A performed as technical triplicates (n = 3). Data are depicted as mean (circles) including standard deviation (error bars) using Origin 2022b. c, Thermostability of WT CtTrl1-LIG and the ΔCTD detected with changes in protein intrinsic fluorescence plotted as the first derivative of the ratio between fluorescence at 350 and 330 nm with temperature. Melting peak was detected at 58.5 °C for WT and 56.7 °C for ΔCTD variant. d, Electrophoretic mobility shift assay (EMSA) with CtTrl1-LIG WT, K148N (both 0–10 µM) and ΔCTD variants (0–10 and 100 µM). Increasing amounts of protein were incubated with the ASL-4U-5C RNA (1 µM) and analyzed by native PAGE using SYBRGold RNA staining. Formation of the LIG•RNA complex was assessed by band shift. This assay was performed in triplicates (n = 3). e, Time course of RNA (2′-P and 3′-OH on the 5′ exon end and 5′-P on the 3′ exon end) adenylylation by CtTrl1-LIG WT. The autoradiogram shows formation of the adenylylated AMP-ASL-4U intermediate via αP32-AMP incorporation. f, Densitometric quantification of the experiment in (e) performed as triplicates (n = 3). Data are depicted as mean (circles) including standard deviation (error bars) using Origin 2022b.The graph reveals a bell-shaped curve for the formation of the AppRNA intermediate over time and its fading through the ligation reaction. g, Time course of RNA (2′-OH and 3′-P on the 5′ exon end and 5′-P on the 3′ exon end) adenylylation by CtTrl1-LIG WT. The autoradiogram shows formation of the adenylylated AMP-ASL-4U intermediate via αP32-AMP incorporation. h, Densitometric quantification of the experiment in (g) performed as triplicates (n = 3). Data are depicted as mean (circles) including standard deviation (error bars) using Origin 2022b.The graph reveals a saturation curve for the formation of the AppRNA intermediate over time.
