Extended Data Fig. 1: Structures of TSEN cleaved pre-tRNAs, circularization assay substrate and RNA stem-loop structure determine CtTrl1-LIG activity.
From: Structure of fungal tRNA ligase Trl1 with RNA reveals conserved substrate-binding principles
a, Yeast tRNA anticodon stem-loop structures. Overview of the anticodon stem-loop regions of all intron-containing tRNAs in yeast. The 5′ exon is colored in purple and the 3′ exon in pink. The sequences for the individual tRNA anticodon stem loops are depicted with the corresponding anticodon in yellow letters. b, Scheme of the ASL-4U RNA substrate for circularization, adenylylation and binding assays. The RNA is derived from the TSEN-cleaved yeast tRNAPhe anticodon stem-loop (ASL) with both strands linked by four uridines (4U). c, Circularization assay of WT CtTrl1-LIG (0.1 µM) with structured and unstructured RNA substrates. Ligation of the linear ASL-4U substrates and the ASL-4U substrate with flipped ends (l) to the circular form (c) was monitored by urea PAGE. d, Circularization assay of WT CtTrl1-LIG (1 µM) with structured and unstructured RNA substrates. Ligation of the linear ASL-4U substrates (l) to the circular form (c) was monitored by urea PAGE. The bracket marks formed concatemers (concat.). e, EMSA with WT CtTrl1-LIG (0–10 µM) and ASL-4U or ASL-4U-6nt (1 µM). Increasing amounts of proteins were incubated with the RNA and analyzed by native PAGE. Formation of the LIG•RNA complex was assessed by band shift. All assays were performed in triplicates (n = 3).
