tRNATyr-GUA-1-1 is pseudouridylated by TruD in vivo and in vitro. (A) Cloverleaf structure of T. thermophilus tRNATyr-GUA-1-1 transcript with the sequence in red containing the TruD recognition sequence. (B and C) Normalized mutation signals at each nucleotide position in tRNATyr-GUA for (B) WT and (C) the ΔtruD strain. (D) Secondary structure of tRNATyr is shown. The red arrow shows the RT primer-annealing region. (E) tRNA mixture was treated with CMC to probe pseudouridine modifications. The resulting RNAs were used in primer extension reactions. The RT product was analyzed using 15% denaturing PAGE, and RT-terminations were visualized by a phosphor imager. The experiment was independently repeated three times (n = 3). A representative gel image of primer extension experiments is presented. (F–I) In vitro pseudouridylation assays with (plus TruD) or without (minus TruD) recombinant TruD using (F, G) tRNATyr-GUA or (H, I) mutant tRNATyr-GUA U35C transcripts. These experiments were independently repeated three times (n = 3) and gave the same results. Of the three trials, one representative chromatogram was shown.
