The presence of pseudouridine in the anticodon alters the genetic code: A possible mechanism for assignment of the AAA lysine codon as asparagine in echinoderm mitochondria

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) tRNAAsn with the anticodon GUU. Nucleotide sequencing of starfish mt tRNAAsn revealed that the anticodon is GΨU, U35 at the anticodon second position being modified to pseudouridine (Ψ). In contrast, mt tRNALys, corresponding to another lysine codon, AAG, has the anticodon CUU. mt tRNAs possessing anti-codons closely related to that of tRNAAsn, but responsible for decoding only two codons each—tRNAHis, tRNAAsp and tRNATyr—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 tRNAAla 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 tRNAAlaGΨU than tRNAAlaGUU. These findings imply that Ψ35 of echinoderm mt tRNAAsn actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria.