tRNA pseudouridine synthase D (TruD) from Thermus thermophilus modifies U13 in tRNA^Asp, tRNA^Glu, and tRNA^Gln and U35 in tRNA^Tyr

The recombinant TruD pseudouridylates substrate tRNAs. (A) Purified T. thermophilus TruD was analyzed by 15% SDS-PAGE and stained with CBB. (B–D) In vitro pseudouridylation assays with (plus TruD) or without (minus TruD) recombinant TruD using in vitro synthesized (B) E. coli tRNA^Glu-UUC, (C) T. thermophilus tRNA^Asp-GUC-1-1, and (D) mutant tRNA^Asp-GUC-1-1 U13C. Pseudouridine formation was detected by nucleoside analysis using HPLC, where pseudouridine eluted ∼5 min. These experiments were performed independently three times (n = 3) and gave the same results; one representative chromatogram is shown.