Fig. 6: 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, Adenylyltransferase assay of CtTrl1-LIG WT, K148N and ΔCTD variants. Top: the autoradiogram after incubation of increasing protein concentrations (0.1–10 µM) with [α32P]ATP. Bottom: the SDS–PAGE was stained with Coomassie brilliant blue (CBB) as protein loading control. This assay was performed in duplicate (n = 2). b, EMSA with WT CtTrl1-LIG and the K148N (both 0–10 µM) and ΔCTD (0–100 µM) variants. Increasing amounts of protein were incubated with the ASL-4U 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 triplicate (n = 3). c, RNA adenylylation by WT CtTrl1-LIG and the K148N and ΔCTD variants (1 µM). The autoradiogram shows formation of the adenylylated ASL-4U intermediate via [α32P]AMP incorporation over time. The depicted gel represents the final result after validation of the assay in triplicate (Extended Data Fig. 8e, n = 3). d, Circularization assay of WT CtTrl1-LIG and the K148N and ΔCTD variants (1 µM). Ligation of the linear ASL-4U substrate (l) to the circular form (c) was monitored by urea PAGE. This assay was performed in triplicate (n = 3).
