Fig. 2: Productive tRNAIle binding requires conformational movements at ScIleRS C-terminal domains.
a The C-ter B domain of ScIleRS is stabilized and binds to the elbow of tRNAIle(GAU). b Interactions with the C-ter B domain induce a conformation of the U20 nucleotide of ScIleRS-bound tRNAIle(GAU) opposite to that of SaIleRS-bound tRNAIle(GAU) (PDB ID: 1FFY). c ScIleRS proteins with mutations located far from the active site exhibited similar or comparable activity to that of the wild-type protein in the tRNA-independent pre-transfer editing assay. Data are presented as means ± SD (n = 3 independent experiments). d Most ScIleRS variants partially or completely lost the tRNAIle isoleucylation activity as measured by tRNA-dependent ATP consumption assay. EctRNAIle(GAU) overexpressed in E. coli cells was utilized in this assay. Data are presented as means ± SD (n = 3 independent experiments). e The EMSA assay result revealed that tRNAIle binding ability of ScIleRSΔCB is weaker than that of wild-type ScIleRS. The in vitro transcript of tRNAIle(GAU) was used in this assay. Similar results were observed in two independent experiments. f The aminoacylation activity of ScIleRS against in vitro transcribed tRNAIle(GAU) and its variants with G19C, G18C&C56A or U55A mutations. All the variants presented significantly lower isoleucylation than that of the wild-type tRNAIle(GAU). Data are presented as means ± SD (n = 3 independent experiments). g, h Structural comparison of the tRNAIle(GAU)-bound ScIleRS with the tRNA-free ScIleRS (PDB ID: 7D5C, colored in gray) (g) and apo ScIleRS (AlphaFold DB: AF-P09436-F1, colored in pink) (h) indicated the conformational changes in the C-terminal domains and ABD of ScIleRS upon tRNAIle binding.
