Larry Simpson

University of California, Los Angeles, Microbiology, Immunology and Molecular Genetics, Emeritus

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Selective importation of RNA into isolated mitochondria from Leishmania tarentolae

RNA, Jul 1, 2000

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from th... more All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA Ile (UAU) and tRNA Gln (CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importa- tion, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA Asp was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than ;17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondrialocalized tRNA Ile as compared to the level of importation of the mainly cytosol-localized tRNA Gln . Furthermore, exchanging the D-arm between the tRNA Ile and the tRNA Gln resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

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Larry Simpson

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