FIG. 1.
The tandem tRNASer-tRNALeu is imported into isolated T. brucei mitochondria. (A) Schematic of the organization of the T. brucei tRNASer and tRNALeu genes and the tRNA substrates transcribed in vitro with T7 RNA polymerase in the presence of [α-32P]UTP for uniform labeling. (B) A 100-μl standard import assay mixture contained 5 × 104 cpm of labeled substrate and isolated mitochondria from 3 × 108 cells. Following import, the mitochondria were collected by centrifugation and resuspended in 25 μl of SME-20 containing 5 mM CaCl2 and 100 mM KCl. Proteinase K was added to a final concentration of 30 μg/ml and the mixture was incubated for 10 min; then 1 volume of SME-20 and 4 mM PMSF were added. The mitochondria were centrifuged, and the pellet was resuspended in 0.3 M sodium acetate–0.5% SDS and phenol-chloroform extracted. The RNAs were collected by precipitation in the presence of 95% ethanol (lanes 1, 4, 7, and 10). In the remaining reactions, proteinase K digestion preceded the addition of 10 U of micrococcal nuclease followed by 10 mM EGTA, lysis of the mitochondria, phenol-chloroform extraction, and ethanol precipitation of the RNAs (lanes 2, 5, 8, and 11). Other reaction mixtures were first treated with 2% CHAPS before proteinase K and micrococcal nuclease treatment (lanes 3, 6, 9, and 12). The isolated RNAs were run on a 6% polyacrylamide–8 M urea gel and visualized by autoradiography.