It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNA(Asn) with the anticodon GUU. Nucleotide sequencing of starfish mt tRNA(Asn) revealed that the anticodon is GΨU, U35 at the anticodon second position being modified to pseudouridine (Ψ). In contrast, mt tRNA(LyS), corresponding to another lysine codon, AAG, has the anticodon CUU. mt tRNAs possessing anticodons closely related to that of tRNA(Asn), but responsible for decoding only two codons each - tRNA(His), tRNA(Asp) and tRNA(Tyr) - were found to possess unmodified U35 in all cases, suggesting the importance of Ψ35 for decoding the three codons. Therefore, the decoding capabilities of two synthetic Escherichia coli tRNA(Ala) variants with the anticodon GΨU or GUU were examined using an E. coli in vitro translation system. Both tRNAs could translate not only AAC and AAU with similar efficiency, but also AAA with an efficiency that was ~2-fold higher in the case of tRNA(Ala)GΨU than tRNA(Ala)GUU. These findings imply that Ψ35 of echinoderm mt tRNA(Asn) actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria.
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