FIG. 3.
RIC-mediated import of tRNA into phospholipid vesicles. (A) Import of Leishmania tRNATyr. Import was assayed by RNase protection. Lane 1, BSA-containing vesicles. Lanes 2 to 6 and 9 to 11, RIC-reconstituted vesicles. The complete system (lanes 2 and 10) contained 100 fmol of tRNATyr and 4 mM ATP. Lanes 3 and 9, ATP omitted. Lane 4, ATP replaced by AMPPCP. Lane 5, 0.5% DOC added after import incubation. Lane 6, tRNAGln(CUG) replacing tRNATyr. Lane 11, 50 μM carbonylcyanide m-chlorophenylhydrazone added. Lanes 7 and 8, input tRNATyr (1 fmol) and tRNAGln (2 fmol), respectively. (B) Left, structure of tRNATyr D arm oligoribonucleotide. Positions are numbered as in the intact tRNA sequence (10). The conserved motif is shown in bold. Right, import of D arm derivatives. Lanes 1 and 2, wild type. Lanes 3 to 6 contained mutants G22:C; G22:C, C13:G; A23:U; and A23:U, U12:A, respectively. Lane 1, BSA-containing vesicles. Lanes 2 to 6, RIC-containing vesicles. (C) Interactions between tRNATyr and tRNAIle. RIC-containing vesicles were incubated with high-specific-activity substrate (50 fmol) with or without low-specific-activity effector (5 fmol), and uptake was assayed. Lanes 1 to 3, tRNATyr substrate; lanes 4 to 6, tRNAIle substrate. Lanes 1 and 4, no effector; lane 2, tRNAIle effector; lanes 3 and 6, tRNAGln(CUG) effector; lane 5, tRNATyr effector. Lanes 7 and 8, input tRNAtyr and tRNAIle, respectively (1 fmol).