A nuclear encoded and mitochondrial imported dicistronic tRNA precursor in Trypanosoma brucei

Abstract

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded and imported into the mitochondrion. A heterogeneous population of RNAs having characteristics of precursor tRNAs have previously been identified within the mitochondrion of T. brucei, suggesting that import occurs via a precursor molecule. In order to identify nuclear genes encoding tRNAs targeted to the mitochondrion, individual mitochondrial tRNAs were separated using two-dimensional gel electrophoresis and enzymatically sequenced. A 1.1-kilobase pair genomic DNA fragment was cloned containing three tRNA genes, tRNA(1)(Ser), tRNA(Leu), and tRNA(2)(Ser). Dicistronic precursors containing the tRNA(1)(Ser) and tRNA(Leu) transcripts with a 59-nucleotide intergenic sequence were identified by reverse transcriptase and polymerase chain reactions and the 5' end of the precursors determined. The dicistronic precursor tRNA is present both in the cytosol and the mitochondrion supporting a model for tRNA import involving precursor tRNA transcripts.