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. CCA found at the 3' end of every tRNA is not transcriptionally encoded, and must be added by tRNA nucleotidyl transferase to the new 3' end produced by 3'-tRNase, respectively. CCA found at the 3' end of every tRNA is not transcriptionally encoded, and must be added by tRNA nucleotidyl transferase into the new 3'end produced by 3'-tRNase. We have investigated the effects of changing sequence and structure on the efficiency and kinetics of Drosophila tRNA/His end processing. RNase P and 3'-tRNase interact differently with the region of universally conserved D/T loop tertiary contacts (Levinger et al., JBC 273:1015, 1998). The 3' end CCA is an anti-determinant for eukaryotic 3'-tRNase (Mohan et al., RNA 5:245, 1999). Sequence and structure in mid-acceptor stem appear to be more important for 3'-tRNase than for RNase P, and 3'- tRNase may contact the 4/69 base pair (Mohan and Levinger, manuscript in preparation). We propose to more completely describe the sequence and structure requirements for pre-tRNA end processing through two types of experiments. First, we wish to saturate the D and T loops of Drosophila tRNA/His with single nucleotide substitutions. We will test the effect of these sequence changes on end processing efficiency and kinetics and on tRNA structure. Second, mutations in human mitochondrial tRNAs have been linked with various inherited diseases. The mutation A7445G in human mitochondrial tRNAs have been linked with various inherited diseases. The mutation A7445G in human mitochondrial tRNA/Ser(UCN) causes non-syndromic deafness (Guan et al., MCB 18:5868, 1998). A7445G, which changes G->UCU to G->CCU which immediately following the 3'-tRNase cleavage site (->), is theorized to caused a 3' end processing defect. We hypothesize that this substitution procedures a 3'- tRNase anti-determinant. Using a mitochondrial tRNA processing extract from cultured human cells and in vitro transcriptions of tRNA precursors, we propose to characterize the postulated processing defect.
Showing the most recent 10 out of 30 publications
