Formation of m1Ψ of H. volcanii tRNA is mediated by TrmY. (A) Nuclease P1 digests of uniformly labeled tRNAs were resolved by 2D-TLC. pA, pC, pG, pU, pΨ, and pm1Ψ indicate 5′-phosphorylated A, C, G, U, Ψ, and m1Ψ, respectively. The radioactive spot corresponding to pm1Ψ is present in both wild-type (H26) and complemented (ΔtrmY+pHTrmY) strains, but is absent from ΔtrmY strain (middle). (B) CMCT-primer extension analyses to determine the modification status of residue at position 54 of H. volcanii elongator tRNAMet were done using primer Met-CCA2 (position marked in Fig. 4A) and total small RNA of wild-type, ΔtrmY, and ΔtrmY+pHTrmY strains. RNAs were treated with (+) or without (−) CMCT for the indicated time (in minutes), followed by alkali (OH-) treatment (+) or no treatment (−). Positions of tRNA residues 54 and 55 are marked on the side. A dark band in CMCT followed by alkali treatment lanes, with an increased intensity in the 20-min treatment lane, indicates the presence of Ψ at that position. These reactions show that unmethylated Ψs are present at position 55 in all three strains, but at position 54 only in the ΔtrmY strain (the band is marked by an arrow).
