Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

Abstract

Annotation of the complete genome of the extreme halophilic archaeon Haloarcula marismortui does not include a tRNA for translation of AUA, the rare codon for isoleucine. This is a situation typical for most archaeal genomes sequenced to date. Based on computational analysis, it has been proposed recently that a single intron-containing tRNA gene produces two very similar but functionally different tRNAs by means of alternative splicing; a UGG-decoding tRNA Instead, we have shown that a tRNA, currently annotated as elongator methionine tRNA and containing CAU as the anticodon, is aminoacylated with isoleucine in vivo and that this tRNA represents the missing isoleucine tRNA. Interestingly, this tRNA carries a base modification of C34 in the anticodon different from the well-known lysidine found in eubacteria, which switches the amino acid identity of the tRNA from methionine to isoleucine and its decoding specificity from AUG to AUA. The methods described in this work for the identification of individual tRNAs present in H. marismortui provide the tools necessary for experimentally confirming the presence of any tRNA in a cell and, thereby, to test computational predictions of tRNA genes.

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

“…Aminoacyl∼tRNA_12 is almost completely deacylated within 15 min, whereas deacylation of aminoacyl∼tRNA_34 is incomplete even after 120 min. The half-lives of deacylation of aminoacyl∼tRNA_12 is ∼5 min which is the same as that of Met∼ ; whereas that of aminoacyl∼tRNA_34 is ∼35 min, which is the same as that of [11]. These data, in combination with in vitro re-aminoacylation using purified recombinant H. marismortui MetRS and isoleucyltRNA synthetase (IleRS) , show that tRNA_12 is the elongator methionine , and that tRNA_34 is the minor tRNA Ile (renamed , X representing an unknown modified base).…”

“…One hypothesis stated that the AUA-reading isoleucine tRNA is derived through alternative splicing of a transcript to produce both a tryptophan tRNA and the isoleucine tRNA [51]. However, using acid urea PAGE, we have shown that the minor AUA-decoding isoleucine tRNA in H. marismortui and other archaeal species is derived from a CAU anticodon-containing tRNA, currently annotated as methionine tRNA, in which C34 in the anticodon is post-transcriptionally modified ( Figure 6A) [11]. In addition, we show that the post-transcriptional modification of C at position 34 in the anticodon of this tRNA, responsible for the switch in amino acid and decoding specificity, is different from those present at position 34 of isoleucine tRNA species in eubacteria and in eukaryotes [52,53].…”

“…various Halobacterial species [11]; Methanocaldococcus jannaschii; Methanococcus maripaludis [12]), yeast (Saccharomyces cerevisiae [13,14]), and mammalian cells (e.g. COS1, HEK293, HeLa cells [15][16][17][18]).…”

Convergent evolution of AUA decoding in bacteria and archaea

“…The presence of a modified C (C*) with an unknown structure was first reported in Haloferax volcanii tRNA Ile , 14 and was also found in Haloarcula marismortui tRNA Ile . 15 Several analytical methods were used to determine the atomic composition and chemical structure of C* isolated from H. marismortui (Euryarchaeota) and Sulfolobus tokodaii (Crenarchaeota), which results in its identification as 2-agmatinylcytidine (agm 2 ( Fig. 1AB).…”

“…We performed a metabolic labeling experiment to test for the incorporation of [ 13 C, 15 N]-labeled agmatine into agm 2 C of tRNA Ile in S. tokodaii and M. acetivorans, and found that agmatine supplied in the medium was efficiently incorporated and used as a substrate for agm 2 C formation. 16 Agmatine is produced from arginine (Arg) via decarboxylation.…”