HUMAN MITOCHONDRIAL 3' END PROCESSING AND DISEASE

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DESCRIPTION (provided by applicant): Eukaryotic tRNAs are transcribed as precursors. The 5? end leader and 3? end trailer are endonucleolytically removed by RNase P and by 3?-tRNase, respectively. 3? end CCA is not transcriptionally encoded, and must be added by tRNA nucleotidyltransferase to the 3? end produced by 3?- tRNase. 3? end CCA, an anti-determinant for eukaryotic 3?-tRNase (Mohan et al., RNA 5: 245,1999), prevents mature tRNA from cycling through 3?-tRNase as a substrate or as an inhibitor, and substitutions in mid-acceptor stem interfere with 3?-tRNase processing (Mohan and Levinger, JMB 303: 605, 2000). The A7445G mutation in human mitochondrial tRNASer(UCN), which causes non-syndromic deafness (Guan et al., MCB 18: 5868, 1998), changes G I UCU--- to G I CCU--- immediately following the 3?-tRNase cleavage site (I), and may produce a 3?-tRNase anti-determinant. Using a human cell culture mitochondrial extract and tRNA precursors produced in vitro, the candidate proposes to characterize the postulated 3? end processing defect and the change(s) in tRNA structure which accompany this and five other substitutions in tRNASer(UCN) linked with human mitochondrial disease, two of which also cause nonsyndromic deafness. In addition, he will study the effects of substitutions in mitochondrial tRNAIle and tRNALeu(UUR) on tRNA structure and 3?-tRNase processing.