Characterization of the lysyl-trna synthetases from trupanosoma brucei

Resumo

Cytosolic and mitochondrial translation systems are separately maintained in eukaryotes. In the specific case of Trypanosoma brucei mitochondrial translation system, aminoacyl¿tRNA synthetases and tRNAs are encoded in the nucleus and their transit through the cytosol on their way to the mitochondria is necessary. The T. brucei genome codes for two tRNALys isoacceptors: tRNALysUUU and tRNALysCUU that are shared by the two translation machineries mentioned. Over the course of this work, it has been determined that although cytosolic and mitochondrial tRNALysUUU populations are transcribed from the same nuclear gene, they present different features. The implications of those features in tRNA mitochondrial import are discussed. The T. brucei genome codes for two proteins with predicted lysyl¿tRNA synthetase activity, namely TbKRS¿1 and TbKRS¿2. Unlike other known KRSs, TbKRS¿2 contains an intriguing carboxy¿terminal extension with no homology to any reported protein. TbKRS¿1 and TbKRS¿2 aminoacylation activity has been analyzed both in vivo and in vitro, their functional localization has been elucidated and the role of the carboxy¿terminal extension on TbKRS¿2 activity has been tested. Furthermore, TbKRS¿2 mitochondrial import determinants and processing events undergone by TbKRS¿2 upon mitochondrial import have been determined. Altogether the results obtained in this work reveal a novel mechanism which ensures the segregation of lysyl¿tRNA synthetase activities in T. brucei. This mechanism involves an extra domain in TbKRS¿2 that prevents the enzyme from being active in the cytosol.