Abstract
Variations in the genetic code are found frequently in mitochondrial decoding systems. Four non-universal genetic codes are employed in ascidian mitochondria: AUA for Met, UGA for Trp, and AGA/AGG(AGR) for Gly. To clarify the decoding mechanism for the non-universal genetic codes, we isolated and analyzed mitochondrial tRNAs for Trp, Met, and Gly from an ascidian, Halocynthia roretzi. Mass spectrometric analysis identified 5-taurinomethyluridine (τm(5)U) at the anticodon wobble positions of tRNA(Met)(AUR), tRNA(Trp)(UGR), and tRNA(Gly)(AGR), suggesting that τm(5)U plays a critical role in the accurate deciphering of all four non-universal codes by preventing the misreading of pyrimidine-ending near-cognate codons (NNY) in their respective family boxes. Acquisition of the wobble modification appears to be a prerequisite for the genetic code alteration.
Highlights
Variations in the genetic code are found frequently in mitochondrial decoding systems
For tRNAGly(AGR) [16], tRNAGly(GGN) [16] and tRNAMet(AUR) [14], positions for pseudouridine were determined biochemically by the post-labeling method [15]. These experiments have identified m5(s2)U at the wobble positions of ascidian mt tRNAs. m5(s2)U was first identified in human and bovine mt tRNAs [23, 24], and has subsequently been found in fish
m5(s2)U appears to be conserved in vertebrate mt tRNAs [23]. m5(s2)U was not found in yeast and Caenorhabditis elegans mt tRNAs [25,26,27], which instead used cmnm5(s2)U as the wobble modification
Summary
Isolation of mt tRNAs—Two hundred grams of the commercially available edible muscle of an ascidian, H. roretzi, was minced, and the tRNA fraction was obtained by phenol extraction as described previously [17]. Two thousand A260 units of crude tRNA were separated by the DEAE-cellulose column. For isolating individual mt tRNAs, the following 5Ј-EC amino-modified DNA probes (Sigma-Aldrich) were designed using “Racess” software [18] and used for reciprocal circulating chromatography [19]: 5Ј-TGGTGCATCAAAGGGACCAACC-CTAACTTA-3Ј for tRNAGly(AGR), 5Ј-CCCACAAACTTTTTCATAGTTTACTGTTGT-3Ј for tRNAMet(AUR), 5Ј-TGGCAGGAAATATATATAATCATTCTATATAACTACT-3Ј for tRNATrp(UGR), 5Ј-TGGTACTACGTCCCTAAAGGATCCCATGAT-3Ј for tRNAGly(GGN), and 5Ј-AAAGAAATAAACTTTCTGGTTTATATTAACCTATATTACC-3Ј for tRNAMet(AUG). Reciprocal circulating chromatography was performed with five-tip columns under the following conditions: the binding of tRNA to the tip columns was performed at 66 °C with binding buffer (1.2 M NaCl, 30 mM HEPES-KOH (pH 7.5), 15 mM EDTA, and 1 mM DTT). The percent frequencies of modifications were calculated by the ratio of the mass chromatogram peak heights for RNA fragments containing modified or unmodified bases
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