An unusual tRNAThr derived from tRNAHis reassigns in yeast mitochondria the CUN codons to threonine

Abstract

The standard genetic code is used by most living organisms, yet deviations have been observed in many genomes, suggesting that the genetic code has been evolving. In certain yeast mitochondria, CUN codons are reassigned from leucine to threonine, which requires an unusual tRNAThr with an enlarged 8-nt anticodon loop (). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that evolved from yeast mitochondrial tRNAHis. To understand this tRNA identity change, we performed mutational and biochemical experiments. We show that Saccharomyces cerevisiae mitochondrial threonyl-tRNA synthetase (MST1) could attach threonine to both and the regular , but not to the wild-type tRNAHis. A loss of the first nucleotide (G−1) in tRNAHis converts it to a substrate for MST1 with a Km value (0.7 μM) comparable to that of (0.3 μM), and addition of G−1 to allows efficient histidylation by histidyl-tRNA synthetase. We also show that MST1 from Candida albicans, a yeast in which CUN codons remain assigned to leucine, could not threonylate , suggesting that MST1 has coevolved with . Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment.

The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

“…Compromising the high selectivity of yeast mitochondrial phenylalanyltRNA synthetase for amino acids leads to a complete loss of mitochondrial respiration (5). The post-transfer editing domain of ThrRS appears to be lost or dysfunctional in yeast mitochondria and mycoplasma (4,35), raising the question as to whether this results in promiscuous translation. We show here that MST1 poorly discriminates against Ser and employs a pre-transfer editing mechanism to remove Ser-AMP (Table 1 and Fig.…”

“…1). The overall editing rate of MST1 is 3-fold faster than the steady-state aminoacylation rate (35), suggesting that the pre-transfer editing activity is important and physiologically relevant in reducing the amount of formed Ser-tRNA Thr . It is also plausible that an unidentified trans-editing factor hydrolyzes Ser-tRNA Thr or that Ser is simply misincorporated at Thr codons at a high frequency in yeast mitochondria, thus decreasing the overall translational fidelity.…”

“…Yeast MST1 is homologous to bacterial ThrRSs but lacks the N-terminal editing domain that hydrolyzes misacylated Ser-tRNA Thr (35,36), prompting us to investigate the fidelity of MST1 for different nearcognate amino acids such as Ser, Val, Ala, and Cys. We first measured the activation rates of Thr and Ser by MST1 using a pyrophosphate exchange assay.…”

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

“…4) (17). MST1 has also evolved since the split between Candida and Saccharomyces to recognize both mitochondrial tRNA Thr species.…”

How tRNAs dictate nuclear codon reassignments: Only a few can capture non-cognate codons

“…To our knowledge, this scenario has been observed in nature only in mitochondria of Saccharomycetaceae where the tRNA Thr UAG and a dedicated ThrRS emerged. 43,44 The tRNA In principle, codon capture could also be achieved by mutations in only the aaRS that would then charge non-cognate tRNAs. However, this would affect all isoacceptor tRNAs and might erase one of the amino acids completely.…”