Identity elements for the aminoacylation of metazoan mitochondrial tRNA(Arg) have been widely conserved throughout evolution and ensure the fidelity of the AGR codon reassignment.

Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNA(Ser)UCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distinct from those possessed by the cytoplasmic tRNAArg in which the major identity elements permit the arginylation of all 5 encoded isoacceptors. We have performed comparative in vitro aminoacylation using an insect mitochondrial arginyl-tRNA synthetase and tRNAArgUCG structural variants. The established identity elements are sufficient to maintain the fidelity of tRNASerUCU reassignment. tRNAs having a UCU anticodon cannot be arginylated but can be converted to arginine acceptance by identity element transplantation. We have examined the evolutionary distribution and functionality of these tRNA elements within metazoan taxa. We conclude that the identity elements that have evolved for the recognition of mitochondrial tRNAArgUCG by the nuclear encoded mitochondrial arginyl-tRNA synthetases of eumetazoans have been extensively, but not universally conserved, throughout this clade. They ensure that the AGR codon reassignment in eumetazoan mitochondria is not compromised by misaminoacylation. In contrast, in other metazoans, such as Porifera, whose mitochondrial translation is dictated by the universal genetic code, recognition of the 2 encoded tRNAArgUCG/UCU isoacceptors is achieved through structural features that resemble those employed by the yeast cytoplasmic system.

Amino acid discrimination by the nuclear encoded mitochondrial arginyl-tRNA synthetase of the larva of a bruchid beetle (Caryedes brasiliensis) from northwestern Costa Rica.

L-canavanine, the toxic guanidinooxy analogue of L-arginine, is the product of plant secondary metabolism. The need for a detoxifying mechanism for the producer plant is self-evident but the larvae of the bruchid beetle Caryedes brasiliensis, that is itself a non-producer, have specialized in feeding on the Lcanavanine-containing seeds of Dioclea megacarpa. The evolution of a seed predator that can imitate the enzymatic abilities of the host permits us to address the question of whether the same problem of amino acid recognition in two different kingdoms has been solved by the same mechanism. A discriminating arginyl-tRNA synthetase, detected in a crude C. brasiliensis larval extract, was proposed to be responsible for insect's ability to survive the diet of L-canavanine (Rosenthal, G. A., Dahlman, D. L., and Janzen, D. H. (1976) A novel means for dealing with L-canavanine, a toxic metabolite. Science 192, 256e258). Since the arginyl-tRNA synthetase of at least three genetic compartments (insect cytoplasmic, insect mitochondrial and insect gut microflora) may participate in conferring L-canavanine resistance, we investigated whether the nuclear-encoded C. brasiliensis mitochondrial arginyl-tRNA synthetase plays a role in this discrimination. Steady state kinetics of the cloned, recombinant enzyme have revealed and quantified an amino acid discriminating potential of the mitochondrial enzyme that is sufficient to account for the overall L-canavanine misincorporation rate observed in vivo. As in the cytoplasmic enzyme of the L-canavanine producer plant, the mitochondrial arginyl-tRNA synthetases from a specialist seed predator relies on a kinetic discrimination that prevents L-canavanine misincorporation into proteins.

The absence of A-to-I editing in the anticodon of plant cytoplasmic tRNA (Arg) ACG demands a relaxation of the wobble decoding rules.

It is a prevalent concept that, in line with the Wobble Hypothesis, those tRNAs having an adenosine in the first position of the anticodon become modified to an inosine at this position. Sequencing the cDNA derived from the gene coding for cytoplasmic tRNA (Arg) ACG from several higher plants as well as mass spectrometric analysis of the isoacceptor has revealed that for this kingdom an unmodified A in the wobble position of the anticodon is the rule rather than the exception. In vitro translation shows that in the plant system the absence of inosine in the wobble position of tRNA (Arg) does not prevent decoding. This isoacceptor belongs to the class of tRNA that is imported from the cytoplasm into the mitochondria of higher plants. Previous studies on the mitochondrial tRNA pool have demonstrated the existence of tRNA (Arg) ICG in this organelle. In moss the mitochondrial encoded distinct tRNA (Arg) ACG isoacceptor possesses the I34 modification. The implication is that for mitochondrial protein biosynthesis A-to-I editing is necessary and occurs by a mitochondrion-specific deaminase after import of the unmodified nuclear encoded tRNA (Arg) ACG.

The influence of identity elements on the aminoacylation of tRNA(Arg) by plant and Escherichia coli arginyl-tRNA synthetases.

Identity elements determine the accurate recognition between tRNAs and aminoacyl-tRNA synthetases. The arginine system from yeast and Escherichia coli has been studied extensively in the past. However, information about the enzymes from higher eukaryotes is limited and plant aminoacyl-tRNA synthetases have been largely ignored in this respect. We have designed in vitro tRNA transcripts, based on the soybean tRNA(Arg) primary structure, aiming to investigate its specific aminoacylation by two recombinant plant arginyl-tRNA synthetases and to compare this with the enzyme from E. coli. Identity elements at positions 20 and 35 in plants parallel those previously established for bacteria. Cryptic identity elements in the plant system that are not revealed within a tRNA(Arg) consensus sequence compiled from isodecoders corresponding to nine distinct cytoplasmic, mitochondrial and plastid isoaccepting sequences are located in the acceptor stem. Additionally, it has been shown that U20a and A38 are essential for a fully efficient cognate E. coli arginylation, whereas, for the plant arginyl-tRNA synthetases, these bases can be replaced by G20a and C38 with full retention of activity. G10, a constituent of the 10:25:45 tertiary interaction, is essential for both plant and E. coli activity. Amino acid recognition in terms of discriminating between arginine and canavanine by the arginyl-tRNA synthetase from both kingdoms may be manipulated by changes at different sites within the tRNA structure.