M. jannaschii TrmY can convert Ψ54 of tRNA to m1Ψ54. (A) Sequences of transcripts used in the reactions. Mutations changing U55 of tRNATrp and U54 of tRNAMet are indicated. The arrow in tRNAMET sequence indicates the position of primer Met-CCA2 used for CMCT-primer extension analyses shown in Figure 3B. (B,C) [α-32P]UTP-labeled tRNATrp was first pseudouridylated by M. jannaschii Pus10 (Mj-Pus10) and then methylated by M. jannaschii TrmY (Mj-TrmY) as described in Materials and Methods. Nuclease P1 or RNase T2 (indicated in panels) digests of purified products were resolved by 2D-TLC on cellulose plates. “p” before or after a nucleoside letter indicates the 5′ or 3′ phosphate of that nucleoside. (Pi) inorganic phosphate. (D) Treatments similar to those in B using [α-32P]UTP-labeled tRNAMet as substrate. The middle panel here (and in B) is shown to indicate that Ψ in the tRNA is produced by Mj-Pus10. (E) TLC separation of RNase T2 digest of [α-32P]CTP-labeled tRNATrp following treatment with Mj-TrmY. (F–I) TLC separation of nuclease P1 digests of [α-32P]UTP-labeled T-arm-Trp, mutant tRNATrp-U55A, tRNATrp-U55G, and tRNATrp-U55C, respectively (indicated in the panels), following treatment with Mj-TrmY.
