Decoding Mechanism of Non-universal Genetic Codes in Loligo bleekeri Mitochondria

Background: Non-universal genetic codes are frequently found in animal mitochondrial decoding systems.

Results: Five mitochondrial tRNAs from Loligo bleekeri were isolated and analyzed by mass spectrometry.

Conclusion: AUA codon is deciphered as Met by CAU anticodon of tRNA^Met(AUR), UGA codon is deciphered as Trp by τm⁵UCA anticodon of tRNA^Trp(UGR).

Significance: AUA codon is deciphered by unmodified CAU anticodon through non-canonical A-C pairing.

Abstract

Non-universal genetic codes are frequently found in animal mitochondrial decoding systems. In squid mitochondria, four codons deviate from the universal genetic code, namely AUA, UGA, and AGA/AGG (AGR) for Met, Trp, and Ser, respectively. To understand the molecular basis for establishing the non-universal genetic code, we isolated and analyzed five mitochondrial tRNAs from a squid, Loligo bleekeri. Primary structures of the isolated tRNAs, including their post-transcriptional modifications, were analyzed by mass spectrometry. tRNA^Met(AUR) possessed an unmodified cytidine at the first position of the anticodon, suggesting that the AUA codon is deciphered by CAU anticodon via non-canonical A-C pairing. We identified 5-taurinomethyluridine (τm⁵U) at the first position of the anticodon in tRNA^Trp(UGR). τm⁵U enables tRNA^Trp to decipher UGR codons as Trp. In addition, 5-taurinomethyl-2-thiouridine (τm⁵s²U) was found in mitochondrial tRNAs for Leu(UUR) and Lys in L. bleekeri. This is the first discovery of τm⁵U and τm⁵s²U in molluscan mitochondrial tRNAs.

Introduction

Variation in the genetic code is a characteristic feature of mitochondrial decoding systems. In animal mitochondria, six codons deviate from the universal genetic code (1). The AUA codon (for Ile) specifies Met in most metazoan mitochondria. The AAA codon (for Lys) changes to assign Asn in echinoderm and some platyhelminth mitochondria. The AGA and AGG (AGR) codons (for Arg) are used for Ser in most invertebrate mitochondria, for Gly in tunicate mitochondria, and as a stop codon in vertebrate mitochondria. The UGA stop codon is used for Trp in all animal mitochondria, and the UAA stop codon is used for Tyr in nematode mitochondria.

To clarify the molecular basis for deciphering the non-universal genetic codes in animal mitochondria, we have been carrying out a project to isolate and analyze individual mitochondrial (mt)³ tRNAs responsible for the non-universal genetic codes in a variety of animal phyla. We have proposed a hypothesis that the acquisition of wobble modifications is a prerequisite for genetic code alteration in mitochondria (2). In bovine mitochondria, we identified and determined the chemical structure of 5-formylcytidine (f⁵C) at the anticodon first (wobble) position of tRNA^Met (3). f⁵C is required to decipher the AUA codon as Met in mitochondria of mammals (3, 4), nematode (Ascaris suum) (5) and insect (Drosophila melanogaster) (6). In addition, we discovered 5-taurinomethyluridine (τm⁵U) and its 2-thio derivative (τm⁵s²U) at the wobble position of human and bovine mt tRNAs responsible for NNR codons (7, 8). Because an unmodified U at the wobble position enables tRNAs to decode any of the four codons in family boxes according to the mitochondrial four-way wobble rule (1, 9), the wobble U in tRNAs responsible for NNR codons must be modified. In fact, τm⁵U is used to assign the UGA codon to Trp in mammals and vertebrates (2, 4). In ascidian (tunicate) mitochondria (10), τm⁵(s²)U is required to decipher all four non-universal genetic codes (AUA for Met, UGA for Trp, and AGR for Gly).

Four non-universal genetic codes exist in the mitochondrial decoding system of the squid Loligo bleekeri, namely, AUA, UGA, and AGA/AGG (AGR) for Met, Trp, and Ser, respectively (Table 1) (11). We previously attempted to elucidate the underlying molecular mechanism that deciphers these non-universal codons by isolating and analyzing mt tRNAs from L. bleekeri. A gene encoding a tRNA^Ser containing a GCT anticodon is encoded in the mtDNA of L. bleekeri. To decode the non-universal AGR codons in addition to the universal AGY codons as Ser, the GCU anticodon of this tRNA must be modified post-transcriptionally. Indeed, we identified 7-methylguanosine (m⁷G) at the wobble position of the mt tRNA^Ser isolated from L. bleekeri liver (12), indicating that all four AGN codons are deciphered by the single tRNA^Ser bearing the m⁷GCU anticodon. The unique four-way wobbling mediated by m⁷G was originally detected in the starfish mitochondrial decoding system (13). In the case of the AUA codon, our group used a postlabeling method to identify a partial f⁵C modification at the wobble position of squid mt tRNA^Met (14), implying that the AUA codon is deciphered by the f⁵CAU anticodon, as observed in mammals, nematode, and insect. However, evidence for this modification relied upon the detection of weak spot with a high background on a two-dimensional thin-layer chromatogram (two-dimensional TLC). To define conclusively the mechanism for deciphering the AUA codon, the frequency of the f⁵C modification needs to be reliably estimated. There have been few studies over the past decade on squid mitochondrial decoding systems because of technical limitations in the isolation and analysis of mt tRNAs. By using the efficient RNA isolation technique based on the reciprocal circulating chromatography (15) and highly sensitive mass spectrometric methods for analyzing RNA molecules (16), we successfully analyzed the post-transcriptional modifications of L. bleekeri mt tRNAs that are responsible for the non-universal genetic codes. Moreover, we discuss our results in the wider context of the evolutionary reorganization of decoding systems in animal mitochondria.

TABLE 1.

Codon table of the squid mitochondrial genetic code

Non-universal genetic codes are shown in bold italics: AUA for Met, UGA for Trp, and AGA/G for Ser. The anticodon sequence of each tRNA gene is shown in parentheses.

EXPERIMENTAL PROCEDURES

Isolation of mt tRNAs

The procedure for isolation of mt tRNAs from edible muscle of L. bleekeri was largely the same as that used for ascidian mt tRNAs reported previously (10). After separation by DEAE-cellulose column chromatography, 2400 A₂₆₀ units of the crude tRNA fraction were isolated from 280 g of squid muscle. Total tRNA (700 A₂₆₀ units) was obtained from 1200 A₂₆₀ units of the crude tRNA fraction by removing contaminating polysaccharides with TRIzol-LS (Invitrogen) followed by rinsing with chloroform. For the isolation of individual mt tRNAs, the following 5′-EC amino-modified DNA probes (Sigma-Aldrich) were designed using Raccess software (17) and used for reciprocal circulating chromatography (RCC) (15): 5′-GGGGTATGAACCCAACAGCTTATTTTTTAGCTTAC-3′ for tRNA^Met(AUR), 5′-TTGAAAGCCTTCAGTTTAACTTAACTTAAAATCTT-3′ for tRNA^Trp(UGR), 5′-AAAGGTAATTAGGAATAAAATAAAGCTGCTAACTT-3′ for tRNA^Ser(AGN), 5′-AAATTCTATGCACTGATCTGCCATCTTAAT-3′ for tRNA^Leu(UUR), and 5′-TCTAGTGCTTACTCATTCGGCCACTTAATA-3′ for tRNA^Lys(AAR). The tDNA sequences were obtained from the NCBI database of L. bleekeri mtDNA (accession number NC_002507) (18, 19). The DNA probes were covalently immobilized on NHS-activated Sepharose 4 Fast Flow (GE Healthcare) and packed into respective tip columns for the RCC instrument. The five tRNA species were simultaneously isolated from 700 A₂₆₀ units of total tRNA by RCC as described (15).

Mass Spectrometric Analysis of mt tRNAs

Each purified tRNA was digested with RNase T₁ and then analyzed by capillary liquid chromatography (LC)/nanoelectrospray ionization mass spectrometry (MS) as described (16). A linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL; Thermo Fisher Scientific) equipped with a custom-made nanospray ion source and a splitless nanoHPLC system (DiNa; KYA Technologies) was employed in this study. Modified bases were assigned by comparing observed m/z values of RNase T₁-digested fragments to the calculated values of these fragments. The bases were allocated on the basis of the sequences of the fragments, which were inferred from the collision-induced dissociation (CID) spectra and assisted by referring to evolutionary conservation of each modified base in tRNAs. N²-methylguanosine (m²G) can be distinguished from 1-methylguanosine (m¹G) by examining the 3′-terminal structure generated by RNase T₁. When RNase T₁ cleaves tRNA on the 3′ side of m²G, a 3′-phosphate is produced. On the other hand, when the 3′ side of m¹G is cleaved by RNase T₁, a 2′,3′-cyclic phosphate (>p) is primarily generated. Mono- or dinucleotide fragments passed through the LC and were not detected. The percent frequencies of modifications were calculated from the ratios of the mass chromatogram peak heights of RNA fragments containing modified bases to those of RNA fragments lacking modified bases.

Nucleoside analysis of tRNAs was performed as described (16, 20). Purified tRNA^Met(AUR) and yeast phenylalanine tRNA (Roche Applied Science) were digested into nucleosides using nuclease P1 (Wako Pure Chemical Industries) and bacterial alkaline phosphatase (BAP.C75; Takara) for 3 h at 37 °C. The digests were then analyzed by a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL).

RESULTS

Five species of squid mt tRNAs were isolated from 700 A₂₆₀ units of total tRNA by RCC, namely, tRNA^Met(AUR), tRNA^Trp(UGR), tRNA^Ser(AGN), tRNA^Leu(UUR), and tRNA^Lys(AAR). Each individual tRNA was faintly detectable on the polyacrylamide gel of the total eluted fraction from the RCC column (Fig. 1). Each band was excised, and tRNA was eluted from the gel pieces for mass spectrometric analysis. Although the final yield of each tRNA could not be precisely measured, we estimated the yield to be within the range of 10–300 fmol.

FIGURE 1.

Isolation of L. bleekeri mt tRNAs by RCC. Isolated L. bleekeri mt tRNAs were resolved by gel electrophoresis on a 10% polyacrylamide gel containing 7 m urea. The gel was stained with SYBR Gold (Invitrogen) and visualized using an FLA-7000 imaging analyzer (Fujifilm). The tRNA bands indicated by arrows were cut out and used for mass spectrometric analysis.

Isolated tRNAs were digested by RNase T₁ and subjected to capillary LC/nanoelectrospray ionization MS to analyze the chemical structures of the modifications. RNA fragments from each tRNA were efficiently separated by capillary LC on the basis of their lengths, base compositions, and sequences (Fig. 2A). Singly and multiply charged anions of RNA fragments were detected with high mass resolution (30,000 units). Modified bases in the fragments were identified on the basis of the precise molecular mass of each RNA fragment, which was calculated by deconvoluting the observed m/z values from the mass spectra (s u p p l e m e n t a l T a b l e 1). Each fragment was further analyzed by an MS/MS experiment using CID to determine the positions of detected modifications within the tRNA sequence (Fig. 2B). The modifications were unambiguously positioned in the sequence by the assignment of y- and c-series product ions (21).

FIGURE 2.

Mass spectrometric analysis of L. bleekeri mt tRNAs. A, base peak chromatograms (BPC) of RNase T₁-digested fragments (upper panels) and mass chromatograms of the anticodon-containing fragments (lower panels) for tRNA^Met(AUR), tRNA^Trp(UGR), tRNA^Ser(AGN), tRNA^Leu(UUR), and tRNA^Lys(AAR) isolated from L. bleekeri. Sequences and molecular masses of the RNA fragments numbered on BPCs are listed in supplemental Table 1. The anticodon sequences are underlined. B, CID spectra of the anticodon-containing fragments from L. bleekeri mt tRNAs. The precursor ions for CID were m/z 1392.69, 1397.17, 1380.41, 1372.16, and 1473.50, respectively. Product ions of the c- and y-series are indicated on the spectra, and the corresponding sequences are indicated. In tRNA^Ser(AGN), a3-B, c3-B, and c4-B represent product ions lacking m⁷G base.

In mt tRNA^Met(AUR), we clearly detected the anticodon-containing fragment (ΨUCAUt⁶ACCCCAAAAAm⁵UGp) bearing N⁶-threonylcarbamoyladenosine (t⁶A) and 5-methyluridine (m⁵U) at positions 37 and 48, respectively (Fig. 2). Although f⁵C has been reported to be partially introduced at the wobble position (14), we could not detect f⁵C at this position (supplemental Fig. 1). This result suggested that the AUA codon is deciphered as Met by the CAU anticodon via non-canonical A-C pairing. We confirmed the presence of 1-methyladenosine (m¹A) at position 9, N²-methylguanosine (m²G) at position 11, and N²,N ²-dimethylguanosine (m²₂G) at position 26 (Fig. 3A and supplemental Table 1). In addition, we newly found a methylated U at position 48 (Fig. 2B), which we determined to be m⁵U by co-injection analysis with authentic m⁵U in total nucleosides of yeast tRNA^Phe (supplemental Fig. 2).

FIGURE 3.

Secondary structures of L. bleekeri mt tRNAs with modifications. A, secondary structures of L. bleekeri mt tRNAs for Met(AUR), Trp(UUR), and Ser(AGN). B, secondary structures of L. bleekeri mt tRNA for Leu(UUR) and Lys(AAR). The numbering system of tRNAs is based on the tRNA database (tRNA db 2009) (36). Pseudouridines were determined previously (12, 14). Frequency of modifications at the wobble position and position 37 is indicated in parentheses.

In mt tRNA^Trp(UGR), we detected the anticodon-containing fragment CUUτm⁵UCAms²i⁶Am¹G>p, which bears τm⁵U at the wobble position, 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A) at position 37, and m¹G at position 38 (Fig. 2). This is the first instance of the detection of τm⁵U in molluscan mt tRNA. The relative frequency of τm⁵U was calculated on the basis of the signal intensity ratio of the modified and unmodified RNA fragments. The wobble base was modified to τm⁵U in 89% of mt tRNA^Trp(UGR), indicating that UGR codons are mainly deciphered by τm⁵UCA anticodons (Fig. 3A), whereas the residual 11% had unmodified U at the wobble position. The hypomodified tRNAs are likely to be the result of their in vivo status rather than different hybridization efficiency with the DNA probes because the length of probes used in this study are long enough to trap all tRNAs with different modification status. In addition, m¹A and m²₂G were found at positions 9 and 26, respectively (Fig. 3A and supplemental Table 1). Moreover, we detected monomethylated uridines (U+me) with unidentified structures at positions 12 and 13 (Fig. 3A and supplemental Table 1).

L. bleekeri mt tRNA^Ser(AGN) contains m⁷G at the wobble position and t⁶A at position 37 (12). The presence of both modifications was confirmed using MS analysis (Figs. 2A and 3A, and supplemental Table 1). m⁷G is distinguishable from the other methylguanosines because RNase T₁ does not cleave the 3′ end of m⁷G. In addition, because the N-glycosyl bond of m⁷G is unstable, the m⁷G base dissociates easily from the RNA fragment upon CID, generating product ions lacking m⁷G base (Fig. 2B). The mass chromatogram showed that the wobble base was completely modified to m⁷G (Fig. 3A).

Having detected τm⁵U in mt tRNA^Trp(UGR), we further analyzed mt tRNA^Leu(UUR) and mt tRNA^Lys(AAR) to identify additional instances of τm⁵U and its derivative in squid mt tRNAs. As expected, the wobble bases of both tRNAs were modified to τm⁵s²U (Fig. 2). The mass chromatograms of the anticodon-containing fragments with different modification statuses (Fig. 3B) showed that 88% of mt tRNA^Leu(UUR) and 90% of mt tRNA^Lys(AAR) contained τm⁵s²U, whereas only 7% of both tRNAs contained τm⁵U. The remaining molecules had a 2-thiouridine or an unmodified U at the wobble position. Other modifications were found in mt tRNA^Leu(UUR) at positions 6 and 10 (m²G) and at positions 9 and 37 (m¹G) (Fig. 3B) and in mt tRNA^Lys(AAR) at position 9 (m¹G), position 10 (m²G), and position 37 (t⁶A) (Fig. 3B). Monomethylated uridines (U+me) with unidentified structures were also found at positions 16, 48, and 52 (Fig. 3B and supplemental Table 1). U+me at position 48 was presumed to be m⁵U as observed in mt tRNA^Met(AUR).

DISCUSSION

We have shown here that L. bleekeri mt tRNA^Met(AUR) possesses an unmodified C at the wobble position (Fig. 3A). Our group previously reported that the wobble position was partially modified to f⁵C in the same tRNA (14). In the study, a weak f⁵C spot along with a strong C spot was detected on the two-dimensional TLC by the postlabeling method, suggesting a false positive. Otherwise, we might use a different strain of L. bleekeri for this study. There might be some individual differences of squid variably expressing a putative tRNA-modifying enzyme for f⁵C formation. Judging from the sensitivity of our MS analysis, if there is only 1% of f⁵C in the anticodon-containing fragment, we can surely detect it. Thus, if a partial f⁵C modification does exist, its frequency must be <1%. In any case, we demonstrated here that a large majority of mt tRNA^Met(AUR) is occupied by unmodified C at the wobble position, indicating that AUA codon is deciphered by CAU anticodon via non-canonical A-C pairing in L. bleekeri mitochondria. This may be a remnant of a primitive decoding system 0that has been used early in evolution, because the domain-specific wobble modifications might have been acquired late during evolution, as suggested by the phylogenetic study on tRNA-modifying enzymes (22).

Some instances of the AUA decoding by a CAU anticodon have been reported in other mitochondrial systems. In D. melanogaster (6), there are two species of mt tRNAs^Met; one has a CAU anticodon with t⁶A at position 37, the other has an f⁵CAU anticodon with an unmodified A at position 37. This indicates that the AUA codon can be read by a CAU anticodon with the assistance of t⁶A37 because t⁶A37 stabilizes tRNA binding to the A site codon (23, 24), or by f⁵CAU without the assistance of t⁶A37. In this study, we detected t⁶A37 in mt tRNA^Met(AUR) (Fig. 3A). The frequency of the t⁶A modification in mt tRNA^Met(AUR) was estimated to be 93% according to the MS data (Fig. 3A). The high modification efficiency indicates the importance of t⁶A37 for the decoding of AUA as Met in L. bleekeri mitochondria. In addition to the anticodon and 3′-adjacent modifications, decoding efficiency can be affected by other regions in tRNAs (25, 26). In the case of Hirsh suppressor tRNA^Trp with G24A and A9C mutations, UGA codon is efficiently deciphered by the CCA anticodon, in which C34 of anticodon pairs with A3 of codon by a non-standard geometry with a single hydrogen bond between N4 imino group of C34 and N1 of A3 (26). These suppressor mutations are considered to enable C34-A3 pairing by facilitating the distortion of tRNA body at A/T state during decoding (26). Thus, there might be some elements in mt tRNA^Met(AUR) that enable CAU anticodon to recognize AUA codon efficiently. In Saccharomyces cerevisiae (27, 28) and mosquito (Aedes albopictus) (29), mt tRNAs^Met are known to have an unmodified CAU anticodon. In S. cerevisiae, the initiator and elongator mt tRNAs^Met have CAU anticodons with m¹G37 and t⁶A37, respectively (27, 28). Collectively, the AUA decoding by a CAU anticodon with the assistance of the modified bases at position 37 might be a general rule.

In animal mitochondria, three strategies are used to decipher AUA codon as Met. The first strategy involves the f⁵CAU anticodon in mt tRNA^Met from mammals, nematode, and Drosophila. Indeed, AUA decoding by f⁵CAU anticodon has been confirmed biochemically (30, 31). The second strategy involves the τm⁵UAU anticodon in ascidian (Halocynthia roretzi) mitochondria. In general, the xm⁵(s²)U modification prevents misreading of near cognate codons ending in pyrimidines (NNY) (9). τm⁵U would enable tRNA to efficiently decode AUR codons as Met. The third strategy involves the unmodified CAU anticodon described in this study. Non-canonical A-C pairing might be involved in AUA decoding by the CAU anticodon, in which t⁶A37 would stabilize the codon-anticodon pairing. Biochemical and structural studies will be required to provide mechanistic insights into this type of decoding. The distribution of different types of AUA decoding in animal mitochondria was assumed to be associated with AUR codon usage. In both human and L. bleekeri mitochondria, AUA codons are used about four times more frequently than AUG codons, indicating that AUA is an abundant codon, whose usage is independent of the type of decoding. Therefore, we were unable to draw any meaningful correlation between AUR codon usage and the types of AUA decoding. The three types of AUA decoding mentioned above rely heavily on the specificities of tRNA-modifying enzymes. In the first type, mt tRNA^Met, containing a CAU anticodon, may be recognized by a putative f⁵C-modifying enzyme that has not been identified yet. In the second type, a τm⁵U-modifying enzyme, probably GTPBP3/MTO1 (4), recognizes mt tRNA^Met with a UAU anticodon. In the third type, although there is no enzyme for f⁵C formation, t⁶A-modifying enzymes (4) recognize mt tRNA^Met with a CAU anticodon and introduce t⁶A at position 37. Further studies directed toward determining the evolutionary distribution of these three types of AUA decoding and characterizing tRNA-modifying enzymes will deepen our understanding of AUA decoding in animal mitochondria.

In L. bleekeri, we identified τm⁵U in mt tRNA^Trp(UGR), and τm⁵s²U in mt tRNA^Leu(UUR) and mt tRNA^Lys(AAR). This is the first reported instance of τm⁵U and τm⁵s²U in molluscan mt tRNAs. τm⁵(s²)U was first identified in mammalian mt tRNAs responsible for Leu(UUR), Trp, Lys, Glu, and Gln (4, 7). Moreover, we also found τm⁵(s²)U in fish,⁴ suggesting that τm⁵(s²)U may be conserved in vertebrate mt tRNAs. In addition, τm⁵(s²)U was found in three mt tRNAs responsible for non-universal genetic codes in ascidian (H. roretzi) mitochondria (10). On the other hand, τm⁵(s²)U was not found in yeast or nematode mt tRNAs, which use 5-carboxymethylaminomethyluridine (cmnm⁵U) instead (32–34). Due to the chemical similarity between cmnm⁵U and τm⁵U, both nucleotides are thought to be synthesized by a similar biosynthetic pathway, which, in the case of τm⁵U, results in the replacement of glycine with taurine (7, 33). To identify the phylogenetic junction at which τm⁵U replaced cmnm⁵U in animal phyla, it will be necessary to analyze additional invertebrate mt tRNAs. Specifically, τm⁵U seems to have emerged in cephalopoda mt tRNAs.

It is intriguing to note that τm⁵s²U is present in L. bleekeri mt tRNA ^Leu(UUR). In general, 2-thiouridine derivatives occur in three tRNA species for Lys, Glu, and Gln. In mammalian mitochondria, τm⁵s²U is found in mt tRNAs for Lys, Glu, and Gln, but not in mt tRNAs for Leu(UUR) or Trp (4). This is because MnmA, a 2-thiouridylase, strictly discriminates the anticodons of tRNAs (35). In human mitochondria, MTU1, a homolog of MnmA, recognizes mt tRNAs for Lys, Glu, and Gln (33). A MTU1 homolog in L. bleekeri may have evolved to recognize mt tRNA ^Leu(UUR).

Another intriguing finding is that m⁵U is present at position 48 in the extra loop of squid mt tRNA^Met(AUR), and probably in mt tRNA^Lys(AAR) as well. In many tRNAs, m⁵U can be found at position 54 in the T-loop, and m⁵U54-methyltransferase is widely distributed across domains of life. Although various modifications occur in the extra loop of many tRNAs, this is the first instance of tRNAs containing m⁵U48. We speculate that a specific methyltransferase for m⁵U48 exists in L. bleekeri and that m⁵U48 is widely distributed in cephalopod and molluscan mt tRNAs. m⁵C is often observed at positions 48 and 49 in the extra loop of tRNAs (36), and m⁵C49 contributes to the structural stabilization of tRNA (37, 38). We speculate that m⁵U48 might have a similar function for the stabilization of tRNAs. In addition, m¹G38 in squid mt tRNA^Trp(UGR) is a unique modification that has never been detected in any other tRNAs to date. m¹G37 is commonly found in many tRNAs, including squid mt tRNA^Leu(UUR). Future studies should examine whether mitochondrial m¹G37-methyltransferase in L. bleekeri has acquired the specificity to introduce m¹G at position 38 in mt tRNA^Trp(UGR). There are still four unidentified methyluridines at positions 12 and 13 in tRNA^Trp(UGR) and positions 16 and 52 in tRNA^Lys(AAR). These modifications might be required for tRNA stabilization as proposed in general (39).

The decoding system for non-universal genetic codes in L. bleekeri mitochondria is summarized in Fig. 4. The AUA codon is deciphered as Met by the CAU anticodon of mt tRNA^Met(AUR), UGA codon is deciphered as Trp by the τm⁵UCA anticodon of mt tRNA^Trp(UGR), and AGR codons are deciphered as Ser by the m⁷GCU anticodon of mt tRNA^Ser(AGN).

FIGURE 4.

Decoding system of the non-universal codons in L. bleekeri mitochondria. Non-universal AUA, UGA, and AGR codons are decoded by mt tRNAs with anticodons CAU, τm⁵UCA, and m⁷GCU, respectively. Non-universal codons are boxed. aa represents amino acid.