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Abstract
The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA(Met) present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA(Met) carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA(Met). In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA(Met). Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA(Met), suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knock-down of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyl-tRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.
Highlights
Mitochondria are of bacterial evolutionary origin, and their translation system is of the bacterial type
Primary Structure of initiation factor 2 (IF2) of T. brucei—Searching the T. brucei genomic data base, we found an open reading frame of 721 aa predicted to encode the orthologue of bacterial IF2
The aim of the present study was to elucidate the role trypanosomal IF2 plays in the mitochondrial translation initiation pathway, in light of the fact that all tRNAs it can use are imported and of eukaryotic type
Summary
Mitochondria are of bacterial evolutionary origin, and their translation system is of the bacterial type. MTF of yeast [6, 7] and bovine mitochondria [8] have been characterized and shown to formylate their respective mitochondria-encoded tRNA substrates. Mitochondrial IF2 of yeast [9] and bovine [10, 11] have been characterized They are similar to the bacterial proteins, the highest homology being found in the GTP-binding domain. A fraction of this tRNA becomes formylated after import [16] This results in the surprising situation in that, T. brucei mitochondria (a typical elongator tRNAMet) functions in translation initiation, provided that it is formylated [17]. The aim of the present study was to characterize mitochondrial IF2 of T. brucei to see whether, as predicted, it was able to interact with the formylated fraction of the imported elongator tRNAMet
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