Abstract
The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNAAla; a G3A change in Ashbya abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.
Highlights
When the genetic code was first deciphered in the 1960s, it was considered to be universal, with all organisms using the same standard code
Bioinformatic analyses suggest that CUU and CUA codons are reassigned from Leu to Ala in mitochondria of A. gossypii
A notable exception is in Kluyveromyces lactis, a close relative of Ashbya. This species avoids CUN codons altogether (Supplementary Table S1), and has no corresponding mitochondrial DNAs (mtDNAs)-encoded tRNA with a UAG anticodon that would allow the recognition of this codon family
Summary
When the genetic code was first deciphered in the 1960s, it was considered to be universal, with all organisms using the same standard code. Single-cell sequencing and biochemical analyses identified yet another, UGA tryptophan-to-glycine reassignment event in SR1 bacteria [5]. These dogma-breaking discoveries suggest that the genetic code is evolvable in nature, and that it could be engineered in synthetic organisms. Conjugative assembly genome engineering have been used to change all 321 known UAG stop codons to the synonymous UAA stop codon. This will enable the abolition of UAG function, thereby permitting subsequent reassignment from ‘stop’ to any natural or non-natural amino acid [7]
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