The D arm of tRNA^Tyr is necessary and sufficient for import into Leishmania mitochondria in vitro

Abstract

Transfer RNAs are selectively imported from the cytoplasm into mitochondria of kinetoplastid protozoa such as Leishmania. The specific structural features of tRNA which determine selectivity are largely unknown. Using an in organello system from Leishmania, the import signals on tRNA^Tyr and on a synthetic transcript which binds to the same receptor, were studied by deletion and reconstruction analyses. In both cases, short oligoribonucleotides (minihelices) containing the sequence UGGYAGAG were imported with high efficiency in the presence of ATP. This motif is present in the D arm of tRNA^Tyr, as well as in the majority of imported Leishmania tRNAs. Deletion of the D arm, or a point mutation in the conserved motif, reduces importability. The import signal coincides with the binding site for the mitochondrial receptor TAB. tRNA^Gln, which is not imported, forms non-productive, TAB-independent complexes with the mitochondrial surface. However, the observation that the imported:bound ratio of the D arm minihelix is higher than that of the entire molecule suggests that the post-binding translocation step is constrained in terms of size or structural flexibility. Kinetic studies of minihelix import indicate stepwise insertion of the molecule into import channels.