Fig. 3: Analysis of the Trl1-LIG RNA-binding interface.
From: Structure of fungal tRNA ligase Trl1 with RNA reveals conserved substrate-binding principles
a, Surface representation of one CtTrl1-LIG molecule with both interacting RNA duplexes in the crystal. The NTD is colored in blue and the CTD in light blue. The RNA strands (bronze and purple; sand and pink) are depicted as cartoons, with the AMP cofactor as sticks in lime. b, Interaction map of CtTrl1-LIG residues with the RNA duplexes from the NTD and the CTD using the same color coding as in a. Essential residues are shown in bold. c, Representation of the exon–exon arrangement extracted from the CtTrl1-LIG–RNA structure. The RNA exon strands are annotated as 5′ exon (purple) and 3′ exon (pink). RNA nucleotides without amino acid contacts are shown in white. d, Zoomed-in view of the CtTrl1-LIG active site with the 3′ end of the 5′ exon (purple; red, oxygen; blue, nitrogen) and the activated 5′ end of the 3′ exon (pink; AMP in lime; red, oxygen; blue, nitrogen) depicted as sticks. The distance of 4.7 Å between the 3′-OH and the 5′-P is indicated by the yellow dashed line. e, Scheme of pre-tRNA ASL coordination. Nucleotide positioning from the crystal structure is shown as sticks in the active site of CtTrl1-LIG (blue). The extending nucleotides are depicted as circles indicating the ASL of a cleaved pre-tRNA (5′ exon in purple and 3′ exon in pink). f, EMSA of WT CtTrl1-LIG and the S168A, H170A, S171A, H182A, S168 H170A and S168 H170A H182A mutants (0–10 µM). Increasing amounts of protein were incubated with the ASL-4U RNA (1 µM) in the presence of AMPcPP 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). g, Fluorescence-based separate strand-ligation assay with WT CtTrl1-LIG and the S168A, H170A, S171A, H182A, S168 H170A and S168 H170A H182A mutants (100 nM). Time-course urea PAGE of the ligation of the 5′ fluorescein (FAM)-labeled 10-nt HAC1 5′ exon oligonucleotide (lower band) with a 20-nt HAC1 3′ exon oligonucleotide was monitored using fluorescence detection. This assay was performed in duplicate (n = 2).
