FIGURE 1.

The tRNA Ψ55 synthase activities of recombinant proteins. Different [α-³²P]CTP-labeled tRNA transcripts (indicated below panels) were incubated with recombinant TRUB1, TRUB2, and i-PUS10 (indicated above panels). Controls were untreated tRNAs. Purified RNA products were digested with RNase T2, and the digests were separated by 2D-TLC and phosphorimaged. As discussed in the text, Ψ in all cases is derived from position 55 of the tRNAs. The mole Ψ/mole tRNA ratio is indicated as a percentage in the panels where Ψ is observed. Representative autoradiograms of repeat experiments are shown. The labels indicate the 3′-monophosphates of corresponding ribonucleosides. Inorganic phosphate is labeled as Pi, which is cropped off in some panels. (A) Treatment of tRNA^Trp and tRNA^Ala. (B) Treatment of U54 and A58 mutants of tRNA^Ala. (C) Treatment of tRNA^iMet and tRNA^AU-Ala. (D) The tRNA^Trp and tRNA^Ala were first treated with TrmI to produce m¹A58 and then treated with the three recombinant proteins.