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Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum

Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum Abstract In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene. tRNA expression from all these genes would generate redundancy in a tRNA that decodes a rare AUA codon. In this study, we investigated the tRNA expression from these genes in L. plantarum and characterized the corresponding tRNAs. The tRNAIle2(CAU) gene products are modified by TilS to produce tRNAIle2(LAU), while tRNAIle2(UAU) lacks modification especially in the anticodon sequence. We found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is not. Our results suggest that the tRNAIle2 redundancy may be related to different roles of these tRNAs in the cell. aminoacylation, mass spectrometry, modification, rare codon, tRNA AUA and AUG codons code for isoleucine and methionine, respectively, and are unique in the universal genetic code. All the other pair of codons ending with a purine (R:A or G) with identical nucleotides at the first and second positions code for the same amino acids (e.g. CUA and CUG code for leucine). This necessitates strict discrimination of the codons for the tRNAs decoding AUA and AUG codons. Bacteria employ two different strategies to cope with this problem. In almost all bacteria, the AUA codon is translated by the tRNAIle harbouring a C*AU anticodon in which the C* indicates a modified nucleobase. In eubacteria, the C is post-transcriptionally modified to lysidine (L). The functional importance and molecular mechanism of the discrimination of AUR codons by the modification has been characterized in Escherichia coli. The significance of the lysidine modification was first demonstrated by an elegant molecular surgery experiment (1). It was shown that the tRNAIle having a non-modified CAU anticodon is not charged with isoleucine but with methionine demonstrating that the lysidine modification not only specifies the codon-anticodon interaction but also affects the tRNA identity. The protein responsible for the lysidine formation is the tRNAIle-lysidine synthetase (TilS) (2). The gene that encodes TilS is essential in E. coli and is also found in almost all the bacterial genomes sequenced. The second strategy is used by bacteria that lack tilS homologues and possess tRNAIle genes with an UAU anticodon (3, 4). Mycoplasma mobile is an example of such species, and the decoding system for AUA codon has been studied recently (3). The UAU anticodon of tRNAIle2 of M. mobile is unmodified, and it has been suggested that ribosomes from M. mobile have acquired the capacity to prevent misreading of the AUG codon by the UAU anticodon. M. mobile is a Mollicutes, a parasitic bacterium. Like other Mollicutes, M. mobile has an extremely small genome of 770 kilobases (5). Mollicutes grow autonomously by maintaining a minimum set of necessary genes (6). The decoding system used for AUA codon by M. mobile enabled loss of tilS gene. We speculate that a third strategy might exist, used by Lactobacillus plantarum for example, in which genes coding for a tilS homologue and tRNAIle with a CAU anticodon exist in addition to the gene that encodes tRNAIle with the UAU anticodon. L. plantarum is a Gram-positive bacterium that is commonly found in starting cultures for food fermentation (7, 8). This bacterium possesses high survival capacity in the human gastrointestinal tract (9) and is likely to have propitious effects on human health (10). The genome of L. plantarum is one of the largest known among lactic acid-metabolizing bacteria (11, 12). This large genome may enable this bacterium to inhabit diverse environmental niches by utilizing a broad range of carbohydrates (12). It is not clear, however, why the bacterium would possess a redundant AUA decoding system. In this study, we used L. plantarum as a model organism to test for the potential coexistence of isoacceptor tRNAs bearing different anticodons that would decode the AUA codon. We found that both tRNAs are indeed expressed in L. plantarum. As expected, the C of the CAU anticodon of tRNAIle2(CAU) is modified to L by the tilS homologue. We found this tRNA to be charged with isoleucine for use in translation. On the contrary, tRNAIle2(UAU) lacks tRNA modifications especially in the anticodon and is not charged with isoleucine. The results suggest that tRNAIle2LAU and tRNAIle2UAU might have different functions in the cell. Materials and Methods Bacterial culture The culture source of L. plantarum WCFS1 was a kind gift from Dr. Ro Osawa (Kobe University). L. plantarum was grown on Man-Rogas-Sharpe (MRS; DifcoTM Lactobacilli MRS broth) agar (containing 1.5% agar, w/v) or cultured statically in MRS broth at 37 °C. RNA preparation L. plantarum total RNA was extracted using ISOGEN II (Nippon Gene) according to manufacturer’s manual with slight modifications. Frozen L. plantarum cells (2 g) were ground in a chilled mortar for 5–10 min. ISOGEN II solution (4 ml) was added to the mortar, and cells were further ground for 10–20 min. The cell suspension was transferred to a tube, and then treated according to the ISOGEN II manual. L. plantarum tRNAIle2(UAU), tRNAIle1(GAU) and tRNAIle2(CAU) were isolated from the extracted total RNA using a solid-phase DNA probe method described in our previous report (13). The 3’-biotinylated DNA probes are described in the Supplementary Table SI. The tDNA sequences were obtained from the Transfer RNA database (http://trna.bioinf.uni-leipzig.de). The isolated tRNA species were analysed on a 10% polyacrylamide/7 M urea gel. Northern hybridization The northern hybridization was performed as reported previously (13). Total RNAs (0.2 A260 units) from L. plantarum and E. coli were separated on a 10% polyacrylamide/7 M urea gel, and then the gel was blotted onto Hybond N+ (GE healthcare). The hybridization was performed at 50 °C for over 12 h. Sequences of the synthetic DNA oligonucleotides for hybridization probes are described in Supplementary Table SI. The DNA probes were 5’-[32 P]-labelled. The hybridized bands were detected using a Typhoon FLA7000 (GE Healthcare). Aminoacylation analyses An aminoacylation assay was performed using the S-100 fraction prepared from L. plantarum and [14 C(U)]-Ile (11.03 GBq/mmol) from Moravek Biochemicals, Inc. Amino acid charging activities were measured by a filter assay using 0.1 A260 units of transcripts for tRNAIle2(UAU) and 0.01 A260 units of purified tRNAIle2(UAU), tRNAIle2(CAU) and tRNAIle2(GAU) (13). The tRNAIle2(UAU) transcript was synthesized from a DNA template obtained by PCR using synthetic DNA oligonucleotides (Supplementary Table SI). Mass spectrometry analyses After purification using the solid-phase DNA probe method, the native tRNAs were desalted by dialysis against water using a nitrocellulose membrane filter (Millipore VSWP02500) and were dried in speed-vac concentrator. The tRNAs were digested 30 min at 37 °C in 5 μl of 20 mM 3-hydroxypicolinic acid (HPA) with 30 units of RNase T1 (Roche) or 5 μl of 100 mM ammonium acetate (pH 7.0) containing 2 µg of RNase A (Roche). The residual cyclic 2', 3'-phosphates formed during RNase T1 digestion were removed by addition of HCl to a final concentration of 0.1 M and incubation at room temperature for 5 min. A 1 μl aliquot of the digest was mixed with 9 µl HPA (40 mg/ml in water: acetonitrile 50 : 50), and 1 µl of the mixture was spotted on the MALDI plate and air-dried (‘dried droplet’ method). MALDI-TOF MS analyses were performed directly on the digestion products using a MALDI-TOF/TOF UltrafleXtreme (Bruker Daltonics). Acquisitions were performed in reflectron positive ion mode. CMCT modification and reverse transcription Pseudouridine (Ψ) was chemically modified by 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT). The chemical modification was performed following the reference (14). Reverse transcription for detection of Ψ was performed using total RNA (0.2 A260 units) from L. plantarum, 5’-32 P-labelled pimer, 1 mM dNTPs and ReverTraAce (Toyobo). Before using for reverse transcription, the total RNA was denatured at 94 °C for 3 min, and then chilled on ice immediately. For detecting nucleotide at position 34, a primer that is complementary to the variable region at the T-loop of L. plantarum tRNAIle2(UAU) was used (Supplementary Table SI). As a control, for detecting Ψ55, a primer sequence complementary to the 18-nt 3’ end sequence of the tRNAIle2(LAU) was used (Supplementary Table SI). DNA sequencing with an fmol DNA Cycle Sequencing System (Promega) was performed using the same primers and the DNA templates for tRNAIle(UAU) or tRNAIle2(LAU) transcription. The oligonucleotide sequences for preparing the DNA templates are described in Supplementary Table SI. Preparation of recombinant tRNAIle-lysidine synthetase The lp_0545 gene in L. plantarum genome has been annotated as tilS (NCBI: http://www.ncbi.nlm.nih.gov). Since one restriction enzyme (XhoI) site exists within the gene, DNA fragment of the lp_0545 gene was amplified by orverlap extension PCR using following primers and KOD FX Neo (TOYOBO) for removing the XhoI site. Primer set 1 is Lp NdeI TilS F, 5’-GGA ATT CCA TAT GAC ACC AAT TCA AAA GTT CAA T-3’ and Lp TilS 805 R, 5’-GCG TCT GCT GCT CTA GCC ACG TCG C-3’. Primer set 2 is Lp TilS 805 F, 5’-GCG ACG TGG CTA GAG CAG CAG ACG C-3’ and Lp XhoI TilS R 5’-CCG CTC GAG ACT CTC GTG TTT TAA GGC TAA AA-3’. Underlines show restriction enzyme sites (NdeI or XhoI). Obtained DNA fragments from primer set 1 and 2 were purified from agarose gel. These two DNA fragments were used as templates for overlap extension PCR using primer set of Lp NdeI TilS F and Lp XhoI TilS R to obtain one fragment with TilS gene with NdeI site at the 5’ end and XhoI site at the 3’ end. The amplified fragment was extracted from the agarose gel and cloned into the NdeI and XhoI sites of the pET22b expression vector. E. coli RosettaTM 2(DE3) was used as the host for the recombinant TilS expression. The C-terminal His-tagged TilS was purified using Ni-SepharoseTM High performance (GE Healthcare). Obtained TilS fraction was concentrated by Amicon Ultra-15 (Millipore). Glycerol was added (final concentration 50%) to the TilS fraction, and the protein was stored at –30 °C. Quantity of the protein was measured with a Bio-Rad protein assay kit (Quick StartTM Bradford Dye Reagent, 1X) using bovine serum albumin as a standard and analysed by 15% SDS-PAGE. Preparation of modified tRNA transcript by TilS The activity of the recombinant TilS was measured at 37 °C as described previously (2). The 50-µl reaction mixture consisted of 100 mM Tris-HCl (pH 7.8), 10 mM KCl, 10 mM MgCl2, 10 mM DTT, 2 mM ATP, 3 µM [14 C(U)]-lysine (11 GBq/mmol, PerkinElmer Life and Analytical Science), 2 µg TilS protein and 0.2 A260 units of tRNA transcript. Synthetic oligo sequences for in vitro tRNAs transcription were described in Supplementary Table SI. We employed two-dimensional TLC for assignment of 14 C-incorporated nucleotide by TilS (15). The 14 C-incorporated tRNA was dissolved in 3 µl of 50 mM ammonium acetate (pH 5.0) and digested with one unit of nuclease P1. A 1-µl aliquot of the sample was spotted onto a thin layer plate (MERCK, TLC Cellulose F; 10 cm x 10 cm) and separated using the solvent systems (a) isobutyric acid, conc. ammonia, water (66: 1: 33, v/v/v), (b) isopropanol, HCl, water (75: 15: 15, v/v/v) and (c) 100 mM sodium phosphate (pH 6.8), ammonium sulphate, 1-propanol (100: 60: 2, v/w/v). Finally, the mass of the modified nucleotide was analysed by MALDI-TOF MS. Results Isolation of L. plantarum tRNAIle species L. plantarum WCFS1 contains five genes that encode isoleucine tRNAs (Transfer RNA Database, http://trna.bioinf.uni-leipzig.de/). One encodes tRNAIle1(GAU), which decodes AUC and AUU codons. Two genes encode tRNAIle2(CAU) (lp_tRNA65, lp_tRNA51), which decodes AUA codons. Although these two genes are annotated as encoding tRNAMet, these anticodons are expected to be modified into LAU by TilS (lp_0545). In addition, there are two genes encoding tRNAIle2(UAU) (lp_tRNA10, lp_tRNA64). The sequences of these two tRNAIle2(UAU) genes differ by only two nucleotides (Fig. 1A). The expression of tRNAIle2(UAU) genes were verified by northern hybridization of total RNAs from L. plantarum and E. coli. A band corresponding to tRNAIle2(UAU) was detected in the L. plantarum total RNA (Supplementary Fig. S1). This result shows that at least one of the tRNAIle2(UAU) genes is expressed. The absence of a band corresponding to tRNAIle2(UAU) in E. coli total RNA confirmed the specificity of the probe used. Sequencing of the RT-PCR products from total RNA from L. plantarum revealed transcripts corresponding to both genes: approximately 75% of the transcripts corresponded to tRNAIle2(UAU)U48, C50 and 25% to tRNAIle2(UAU)C48, U50 (data not shown). We then isolated tRNAIle2(UAU), tRNAIle2(CAU) and tRNAIle1(GAU) from L. plantarum using a solid-phase DNA probe column method (Fig. 1B) (13). The result demonstrates that both tRNAIle2(UAU) and tRNAIle2(CAU) exist as stable species in L. plantarum. Fig. 1 Open in new tabDownload slide Isolation of L. plantarum tRNAIles. (A) Clover leaf structures of L. plantarum tRNAIle2(UAU) and tRNAIle2(CAU), which are products of lp_tRNA10 and lp_tRNA65, respectively. C48 and U50 of tRNAIle2(UAU) are highlighted with numbers. In the other tRNAIle2(UAU) encoded by lp_tRNA64, these positions are U48 and C50. In tRNAIle2(CAU), C34 is modified to L by TilS. Also C20a, U45, U51 and A63, which are substituted by U20a, G45, A51 and U63, respectively, in the other tRNAIle2(CAU) encoded by lp_tRNA51 are highlighted with numbers. (B) Purification of L. plantarum small RNAs and isolation of tRNAIle2(UAU), tRNAIle2(CAU) and tRNAIle1(GAU). Purified RNAs were separated on a 10% polyacrylamide/7 M urea gel and detected by toluidine blue staining. The anticodon of L. plantarum tRNAIle2(CAU) is modified by TilS In most eubacteria, the anticodon of tRNAIle2(CAU) is modified by TilS after transcription. The L modification of the anticodon loop is required for recognition by IleRS and for decoding of AUA codon (16). In the presence of ATP, TilS catalyzes covalent bond formation between the ε-amino group of lysine and C2 of cytosine in the tRNAIle2(CAU) anticodon. The lp_0545 gene of L. plantarum genome is annotated as a tilS gene (11, 17). This gene was cloned into an expression vector, and the recombinant protein was partially purified (Supplementary Fig. S2). TilS activity was measured by the incorporation of [14 C]-lysine into in vitro transcribed tRNAs. In the presence of partially purified recombinant TilS, lysine was incorporated into the tRNAIle2(CAU) transcript, whereas no incorporation was observed for tRNAMet, although this tRNA has the same anticodon sequence as tRNAIle2(CAU) (Fig. 2A). The strict discrimination by TilS between tRNAIle2(CAU) and tRNAMet has been reported for E. coli TilS (16). Alteration of the CAU anticodon of tRNAIle2(CAU) to UAU completely abolished incorporation of lysine by TilS suggesting that the anticodon cytidine is the site of incorporation (Fig. 2A). Fig. 2 Open in new tabDownload slide The C34 of L. plantarum tRNAIle2(CAU) is modified by TilS. (A) Time course of lysidine formation in tRNAIle2(CAU) (circle), tRNAIle2(CAU/UAU) mutant (triangle) and initiator tRNAMet(CAU) (square) catalyzed by TilS. (B) The tRNAIle2(CAU) transcript was incubated with partially purified recombinant TilS, and modified nucleotides were identified by 2 D TLC. Positions of standard markers (pA, pG, pC and pU) are enclosed by dotted circles. (C) Mass spectrum of L. plantarum tRNAIle2(CAU) transcript modified in vitro by purified TilS and digested with RNase A. The spectrum shows the lysidine-34 containing fragment LAUp. Inset spectrum shows the tRNAIle2(CAU) transcript without modification. (D) Detection of lysidine-34 in the native L. plantarum tRNAIle2(LAU) by MALDI-TOF MS. Mass spectrum of the native L. plantarum tRNAIle2(LAU) digested by RNase A shows a lysidine-34 containing fragment LAUp. The spectrum is enlarged around the AACp fragment to show the putative methylation at the position 37. Supplementary Table SII gives theoretical and empirical masses of singly protonated ions derived from the RNase A fragments of the L. plantarum tRNAIle2(LAU). Formation of lysidine was verified first by two-dimensional TLC. The tRNAIle2(CAU) treated with TilS in the presence of [14 C]-lysine was digested with nuclease P1, and the resulting nucleotides were analysed by 2 D TLC (Fig. 2B). The results confirmed the formation of lysidine as shown previously (15). TilS-treated tRNAIle2(CAU) transcript was then digested with pyrimidine-specific RNase A, and samples were analysed by MALDI-TOF MS. Unlike the spectrum of tRNAIle2(CAU) without lysine ligation by TilS (Fig. 2C, inset), a peak at m/z 1087 corresponding to the mass of LAU was observed in the spectrum of tRNAIle2(CAU) treated with TilS (Fig. 2C). Furthermore, the peak of the unique A35U adjacent to the position 34 decreased when L34 was present (data not shown). These results show that modification of cytidine into lysidine prevented cleavage at position 34 by RNase A resulting in the appearance of LAU fragment and a decrease in the amount of the unique AU fragment. From this result, we conclude that C34 is the site of lysidine modification in tRNAIle2(CAU). Finally, we analysed tRNAIle2(CAU) isolated from L. plantarum by MALDI-TOF MS to determine whether lysidine was present. A fragment (m/z 1087) with the mass of LAU was also observed in this sample (Fig. 2D), confirming the lysidine modification in the anticodon in vivo. Hereafter, tRNAIle2(CAU) of L. plantarum will be denoted as tRNAIle2(LAU). We did not detect a fragment at m/z 1126 indicating that the native tRNA does not have N6-threonylcarbamoyladenosine (t6A) at position 37 (a fragment at m/z 1126 with sequence t6AAUp was not detectable); this is in contrast to E. coli tRNAIle(LAU), which does have the t6A modification (18). Instead, the spectrum of L. plantarum tRNAIle2(LAU) shows an ion at m/z 996.27 corresponding to the mass of methylated A37ACp fragment (Fig. 2D, inset). The result suggests the presence of N6-methyladenosine (m6A) at position 37 as is the case for tRNAIle(LAU) from Bacillus subtilis and from Mycoplasma capricolum (19, 20). Characterization of tRNAIle2(UAU) The tRNAIle2(UAU) isolated by solid-phase DNA probe column method was digested with RNase T1 and analysed for post-transcriptional modifications by MALDI-TOF MS. Methylation was detected in the fragment UUCGp. This fragment contains position 54 that is known to be modified into 5-methyluridine in almost all tRNAs (Fig. 3A). No other modifications were observed, even in the anticodon loop, by MALDI-MS (Fig. 3B). In some eukaryotes, AUA codons are decoded by the tRNAIle that has a ΨAΨ anticodon where Ψ stands for pseudouridine (21). Since pseudouridine and uridine have the same molecular mass, these two nucleotides cannot be distinguished by MALDI-MS. In a reverse transcription-based analysis (14), no stops of the reverse transcriptase were observed, indicating that no pseudouridines or other modified nucleotides were present in the anticodon loop of tRNAIle(UAU) (Fig. 4). Fig. 3 Open in new tabDownload slide MALDI mass spectrometry analysis of the native L. plantarum tRNAIle2(UAU). Native L. plantarum tRNAIle2(UAU) was digested with RNase T1 and subjected to MALDI-TOF mass spectrometry. Inserts A and B show regions containing position 54 and the anticodon loop, respectively. Theoretical and measured masses of the resulting fragments are listed in the table. Fig. 4 Open in new tabDownload slide Detection of pseudouridine (Ψ) by CMCT-RT. The regions complementary to primers used for reverse transcription are marked with dashed arrows on the cloverleaves schematics. Primers were specific for tRNAIle2(UAU) or tRNAIle2(LAU). Primer extensions were performed on total RNA from L. plantarum. The total RNAs were treated with CMCT for 2, 10 and 20 min followed or not by alkaline (OH-) treatment (+ or –). Dideoxy DNA sequencing ladders (lanes C, T, A, G) were prepared with the same primers. No pseudouridine in the anticodon of tRNAIle2(UAU) was detected. Of tRNAIle2 variants in L. plantarum, only tRNAIle2(LAU) is charged with isoleucine As both species of tRNAIle2, one with LAU anticodon and another with an unmodified UAU anticodon, exist in L. plantarum, we examined whether both tRNAIle isoforms accept isoleucine. The tRNAIle2(UAU), tRNAIle2(LAU) and tRNAIle1(GAU) isolated from L. plantarum were assayed for aminoacylation with [14 C]-isoleucine in vitro using an L. plantarum S-100 fraction. The tRNAIle1(GAU) was used as a positive control. Charging with isoleucine was observed for tRNAIle2(LAU) and tRNAIle1(GAU) (Fig. 5A). In contrast, tRNAIle2(UAU) was not charged with isoleucine, and the level of radioactivity in this sample was the same as observed without tRNA. Fig. 5 Open in new tabDownload slide Of L. plantarum tRNAIle2 variants, only tRNAIle2(LAU) is charged with isoleucine. (A) Ile charging of native tRNAIle2(LAU) and tRNAIle2(UAU) purified from L. plantarum. Native tRNAIle1(GAU) was used as a positive control. Ile acceptance activities were measured by [14 C]Ile incorporation in the presence of L. plantarum S-100. Error bars are SEM for three independent experiments. (B) Ile charging activities of L. plantarum tRNAIle2 transcript and its anticodon variants. Specificity of isoleucine charging was further tested using in vitro transcribed tRNAIle2(LAU), tRNAIle2(CAU) and a mutant of this tRNA with an UAU anticodon as substrates. tRNA substrates were prepared in vitro as described above. Only tRNAIle2(LAU) was charged with isoleucine (Fig. 5B). The result agrees with a previous report that showed lysidine at wobble position of the anticodon of tRNAIle2 is a critical determinant for charging with isoleucine (1). Less isoleucine was incorporated into the in vitro prepared tRNAIle2(LAU) (approximately 50 pmol/A260 in 10 min) compared to the tRNAIle2(LAU) isolated from cells (Fig. 5A and B). This is likely to be due to the incomplete lysidylation of the transcript (estimated to be 50%) and possible contributions of other modifications in tRNAIle2(LAU), such as the previously described methylation at position 37, to the charging efficiency (22). Discussion AUA is a rarely used codon in most organisms. For example, in E. coli, AUA accounts for less than 0.5% of the 1,356,539 codons used in annotated genes. Our analysis showed that in L. plantarum, AUA codons are used even less frequently than this. Of the 920,850 codons in annotated genes, the AUA codon accounts for less than 0.3%. Within the three isoleucine codons (AUA, AUC, AUU), AUA codons represent less than 5% and about 60% of L. plantarum genes do not contain a single AUA codon (Supplementary Fig. S3). In bacteria, a strong correlation is observed between the frequency of codon usage and the concentration of the tRNA decoding the codon (23). Further, the concentrations of individual tRNAs are proportional to the copy numbers of the respective genes in E. coli and B. subtilis (24–26). Thus, the number of genes coding for tRNAIle2(CAU) and tRNAIle2(UAU) in L. plantarum is disproportionate given how rare the AUA codons are. Here, we showed that two tRNA species, tRNAIle2(CAU) and tRNAIle2(UAU), capable of decoding AUA codons are expressed in L. plantarum. As observed in many other bacterial species, we found that tRNAIle2 with the CAU anticodon is modified into LAU and charged with isoleucine to fulfil its role in translation. The tRNAIle2 with the UAU anticodon was not charged with isoleucine. Sequence elements that are important for IleRS have been identified (16, 22) (Supplementary Fig. S4). L. plantarum tRNAIle2(LAU) possesses all the known IleRS determinants, whereas tRNAsIle2(UAU) is missing some, likely explaining why the former is charged with isoleucine and the latter is not. In the absence of lysidine modification, tRNAIle2(LAU) possesses most positive determinants for recognition by MetRS (27) (Supplementary Fig. S4). Lysidine is a critical negative determinant for MetRS in E. coli (1, 16). Here, the modification of the anticodon of tRNAIle2(LAU) by TilS also plays a crucial role in determining the tRNA identity also in L. plantarum. From the crystal structure of Staphylococcus aureus IleRS in complex with E. coli tRNAIle1 (28) and amino acid sequence comparison of IleRSs from various species, Suzuki and co-workers proposed how different IleRSs recognize LAU or UAU anticodons (3). They reported that Trp890 (numbering for S. aureus IleRS) in the C-terminal Zn-binding domain of IleRS is well conserved in species bearing a tilS homologue. In bacterial species not containing a tilS homologue but having tRNAIle2(UAU), the Trp890 is replaced with arginine (M. mobile), lysine (Mycoplasma haemofelis), or leucine (Mycoplasma suis). Mutation of the tryptophan in E. coli IleRS to arginine results in a 10-fold increase in recognition of tRNAIle2(UAU), while the activity toward tRNAIle2(LAU) is maintained (3). Interestingly, even though IleRS of L. plantarum bears arginine at the corresponding position (Supplementary Fig. S5), the enzyme did not charge the tRNAIle2(CAU/UAU) mutant nor the tRNAIle2(UAU). This suggests that L. plantarum IleRS distinguishes the LAU anticodon from the UAU anticodon by a mechanism that differs from that of the E. coli enzyme. Based on the study of AUA decoding system in M. mobile, the evolution of AUA decoding system has been proposed (3). According to this theory, majority of bacteria now existing are at the stage of evolution where tRNAIle with CAU anticodon is modified into LAU by TilS and the anticodon is recognized specifically by IleRS but not by MetRS. This system ensures fidelity of decoding. From this state, in some bacteria, the tRNAIle(TAT) gene would have been generated by the duplication of the tRNAIle(CAT) gene followed by a C-to-T transition at the wobble position of one gene. In such bacteria, IleRS might have evolved to recognize the UAU anticodon. Once IleRS acquired sufficient recognition of UAU anticodon, some bacteria might have lost tilS as has been observed for M. mobile. In this bacterium, evolution has made ribosomes to be capable of preventing misreading of AUG codon by the UAU anticodon. For L. plantarum the situation seems to be outside of this schema as the sequence of tRNAIle(UAU) differs significantly from that of tRNAIle(LAU) in terms of IleRS recognition determinants and the presence of long variable loop. This may also explain, in part, why tRNAIle(UAU) is not charged with isoleucine and may suggest that this tRNA does not function for canonical AUA decoding. Interestingly, both tRNAIle(TAT) genes are found in uninducible prophage sequences of L. plantarum WCFS1 genome (29) suggesting that these tRNAs derived from phages. Prophages contribute to increase the competitiveness of the bacterial populations in their ecological niches (30). A bioinformatic analysis of metagenomic data revealed that stop-to-sense codon reassignment is extensively found in prophages (31). In the case of phages found in the human oral cavity environments, a noncanonical tRNA in the phage is suspected to be responsible for this codon reassignment. Likewise tRNAIle2(UAU) might be involved in mechanisms that leads to genetic code flexibility (32) that confers L. plantarum high survival ability for various environmental changes. Besides the canonical role in translation, tRNAs and tRNA-like molecules have diverse functions. These include enzymatic modification of proteins leading to signalling for degradation of the labelled proteins, cell envelope modification and synthesis of small molecules such as antibiotics (33). tRNAs can also function as sensor-regulator molecules as in the case of T-box riboswitches (34) or in the stringent response (35). Most of these functions, together with canonical translation, utilize the tRNA’s ability to be charged with amino acids. As mentioned above, we did not observe charging of isoleucine to tRNAIle2(UAU). One of the peculiar feature of tRNAIle2(UAU) is the unique secondary structure with a long variable loop. This is characteristic of class II tRNAs, including leucine and serine tRNAs. We tested charging of tRNAIle2(UAU) with these amino acids, but we did not observe incorporation of these amino acids (data not shown). However, it should be noted that L. plantarum is capable to inhabit diverse environmental niches, therefore it may be possible that charging of tRNAIle2(UAU) occurs in certain conditions and/or the tRNA functions to respond or sense environmental changes. It is also possible that tRNAIle2(UAU) is charged with amino acid other than isoleucine. These possibilities should be clarified. To conclude we showed that tRNAIle2(LAU) and tRNAIle2(UAU) are expressed in L. plantarum creating redundancy in tRNAs that would decode rare AUA codons. However, we found that tRNAIle2 (UAU) is not charged with isoleucine. This contrasts with efficient charging of tRNAIle2 (LAU) with isoleucine to fulfil its role in translation. Altogether, the results suggest that tRNAIle2 (LAU) functions as canonical tRNA, while tRNAIle2 (UAU) might display distinct functions in the cell. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements The authors thank Dr. Ro Osawa (Kobe University) for the gift of L. plantarum WCFS1 strain; and Dr. Takeo Suzuki (the University of Tokyo) and Dr. Ikuko Masuda-Nishimura (Kikkoman Corp.) for their technical suggestions. The authors also thank Dr. Henri Grosjean for useful discussions. Funding This work was supported by Kurata Grants from the Kurata Memorial Hitachi Science and Technology Foundation (to C.T.), by a grant for young researchers from JGC-Scholarship Foundation (to C.T.) by Grants in Aid for Scientific Research from the Japan Society for Promotion of Science (16K18493 to C.T.) and by grants from CNRS PRC 23556 (to S.Y.) and Sumitomo Foundation (101071, 111344 to S.Y.). Funding for open access charges is provided by CNRS funding to S.Y. 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USA 70 , 1564 – 1568 Google Scholar Crossref Search ADS WorldCat Abbreviations Abbreviations CMCT 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate HPA hydroxypicolinic acid IleRS isoleucyl-tRNA synthetase L lysidine m6A N6-methyladenosine MALDI-TOF MS matrix assisted laser desorption ionisation - Time of flight mass spectrometry MetRS methionyl-tRNA synthetase Ψ pseudouridine R purine RT reverse transcription SEM standard error of the mean t6A N6-threonylcarbamoyladenosine TilS tRNAIle-lysidine synthetase TLC thin-layer chromatography © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum

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Oxford University Press
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Copyright © 2022 The Japanese Biochemistry Society
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0021-924X
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10.1093/jb/mvx075
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29092074
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Abstract

Abstract In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene. tRNA expression from all these genes would generate redundancy in a tRNA that decodes a rare AUA codon. In this study, we investigated the tRNA expression from these genes in L. plantarum and characterized the corresponding tRNAs. The tRNAIle2(CAU) gene products are modified by TilS to produce tRNAIle2(LAU), while tRNAIle2(UAU) lacks modification especially in the anticodon sequence. We found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is not. Our results suggest that the tRNAIle2 redundancy may be related to different roles of these tRNAs in the cell. aminoacylation, mass spectrometry, modification, rare codon, tRNA AUA and AUG codons code for isoleucine and methionine, respectively, and are unique in the universal genetic code. All the other pair of codons ending with a purine (R:A or G) with identical nucleotides at the first and second positions code for the same amino acids (e.g. CUA and CUG code for leucine). This necessitates strict discrimination of the codons for the tRNAs decoding AUA and AUG codons. Bacteria employ two different strategies to cope with this problem. In almost all bacteria, the AUA codon is translated by the tRNAIle harbouring a C*AU anticodon in which the C* indicates a modified nucleobase. In eubacteria, the C is post-transcriptionally modified to lysidine (L). The functional importance and molecular mechanism of the discrimination of AUR codons by the modification has been characterized in Escherichia coli. The significance of the lysidine modification was first demonstrated by an elegant molecular surgery experiment (1). It was shown that the tRNAIle having a non-modified CAU anticodon is not charged with isoleucine but with methionine demonstrating that the lysidine modification not only specifies the codon-anticodon interaction but also affects the tRNA identity. The protein responsible for the lysidine formation is the tRNAIle-lysidine synthetase (TilS) (2). The gene that encodes TilS is essential in E. coli and is also found in almost all the bacterial genomes sequenced. The second strategy is used by bacteria that lack tilS homologues and possess tRNAIle genes with an UAU anticodon (3, 4). Mycoplasma mobile is an example of such species, and the decoding system for AUA codon has been studied recently (3). The UAU anticodon of tRNAIle2 of M. mobile is unmodified, and it has been suggested that ribosomes from M. mobile have acquired the capacity to prevent misreading of the AUG codon by the UAU anticodon. M. mobile is a Mollicutes, a parasitic bacterium. Like other Mollicutes, M. mobile has an extremely small genome of 770 kilobases (5). Mollicutes grow autonomously by maintaining a minimum set of necessary genes (6). The decoding system used for AUA codon by M. mobile enabled loss of tilS gene. We speculate that a third strategy might exist, used by Lactobacillus plantarum for example, in which genes coding for a tilS homologue and tRNAIle with a CAU anticodon exist in addition to the gene that encodes tRNAIle with the UAU anticodon. L. plantarum is a Gram-positive bacterium that is commonly found in starting cultures for food fermentation (7, 8). This bacterium possesses high survival capacity in the human gastrointestinal tract (9) and is likely to have propitious effects on human health (10). The genome of L. plantarum is one of the largest known among lactic acid-metabolizing bacteria (11, 12). This large genome may enable this bacterium to inhabit diverse environmental niches by utilizing a broad range of carbohydrates (12). It is not clear, however, why the bacterium would possess a redundant AUA decoding system. In this study, we used L. plantarum as a model organism to test for the potential coexistence of isoacceptor tRNAs bearing different anticodons that would decode the AUA codon. We found that both tRNAs are indeed expressed in L. plantarum. As expected, the C of the CAU anticodon of tRNAIle2(CAU) is modified to L by the tilS homologue. We found this tRNA to be charged with isoleucine for use in translation. On the contrary, tRNAIle2(UAU) lacks tRNA modifications especially in the anticodon and is not charged with isoleucine. The results suggest that tRNAIle2LAU and tRNAIle2UAU might have different functions in the cell. Materials and Methods Bacterial culture The culture source of L. plantarum WCFS1 was a kind gift from Dr. Ro Osawa (Kobe University). L. plantarum was grown on Man-Rogas-Sharpe (MRS; DifcoTM Lactobacilli MRS broth) agar (containing 1.5% agar, w/v) or cultured statically in MRS broth at 37 °C. RNA preparation L. plantarum total RNA was extracted using ISOGEN II (Nippon Gene) according to manufacturer’s manual with slight modifications. Frozen L. plantarum cells (2 g) were ground in a chilled mortar for 5–10 min. ISOGEN II solution (4 ml) was added to the mortar, and cells were further ground for 10–20 min. The cell suspension was transferred to a tube, and then treated according to the ISOGEN II manual. L. plantarum tRNAIle2(UAU), tRNAIle1(GAU) and tRNAIle2(CAU) were isolated from the extracted total RNA using a solid-phase DNA probe method described in our previous report (13). The 3’-biotinylated DNA probes are described in the Supplementary Table SI. The tDNA sequences were obtained from the Transfer RNA database (http://trna.bioinf.uni-leipzig.de). The isolated tRNA species were analysed on a 10% polyacrylamide/7 M urea gel. Northern hybridization The northern hybridization was performed as reported previously (13). Total RNAs (0.2 A260 units) from L. plantarum and E. coli were separated on a 10% polyacrylamide/7 M urea gel, and then the gel was blotted onto Hybond N+ (GE healthcare). The hybridization was performed at 50 °C for over 12 h. Sequences of the synthetic DNA oligonucleotides for hybridization probes are described in Supplementary Table SI. The DNA probes were 5’-[32 P]-labelled. The hybridized bands were detected using a Typhoon FLA7000 (GE Healthcare). Aminoacylation analyses An aminoacylation assay was performed using the S-100 fraction prepared from L. plantarum and [14 C(U)]-Ile (11.03 GBq/mmol) from Moravek Biochemicals, Inc. Amino acid charging activities were measured by a filter assay using 0.1 A260 units of transcripts for tRNAIle2(UAU) and 0.01 A260 units of purified tRNAIle2(UAU), tRNAIle2(CAU) and tRNAIle2(GAU) (13). The tRNAIle2(UAU) transcript was synthesized from a DNA template obtained by PCR using synthetic DNA oligonucleotides (Supplementary Table SI). Mass spectrometry analyses After purification using the solid-phase DNA probe method, the native tRNAs were desalted by dialysis against water using a nitrocellulose membrane filter (Millipore VSWP02500) and were dried in speed-vac concentrator. The tRNAs were digested 30 min at 37 °C in 5 μl of 20 mM 3-hydroxypicolinic acid (HPA) with 30 units of RNase T1 (Roche) or 5 μl of 100 mM ammonium acetate (pH 7.0) containing 2 µg of RNase A (Roche). The residual cyclic 2', 3'-phosphates formed during RNase T1 digestion were removed by addition of HCl to a final concentration of 0.1 M and incubation at room temperature for 5 min. A 1 μl aliquot of the digest was mixed with 9 µl HPA (40 mg/ml in water: acetonitrile 50 : 50), and 1 µl of the mixture was spotted on the MALDI plate and air-dried (‘dried droplet’ method). MALDI-TOF MS analyses were performed directly on the digestion products using a MALDI-TOF/TOF UltrafleXtreme (Bruker Daltonics). Acquisitions were performed in reflectron positive ion mode. CMCT modification and reverse transcription Pseudouridine (Ψ) was chemically modified by 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT). The chemical modification was performed following the reference (14). Reverse transcription for detection of Ψ was performed using total RNA (0.2 A260 units) from L. plantarum, 5’-32 P-labelled pimer, 1 mM dNTPs and ReverTraAce (Toyobo). Before using for reverse transcription, the total RNA was denatured at 94 °C for 3 min, and then chilled on ice immediately. For detecting nucleotide at position 34, a primer that is complementary to the variable region at the T-loop of L. plantarum tRNAIle2(UAU) was used (Supplementary Table SI). As a control, for detecting Ψ55, a primer sequence complementary to the 18-nt 3’ end sequence of the tRNAIle2(LAU) was used (Supplementary Table SI). DNA sequencing with an fmol DNA Cycle Sequencing System (Promega) was performed using the same primers and the DNA templates for tRNAIle(UAU) or tRNAIle2(LAU) transcription. The oligonucleotide sequences for preparing the DNA templates are described in Supplementary Table SI. Preparation of recombinant tRNAIle-lysidine synthetase The lp_0545 gene in L. plantarum genome has been annotated as tilS (NCBI: http://www.ncbi.nlm.nih.gov). Since one restriction enzyme (XhoI) site exists within the gene, DNA fragment of the lp_0545 gene was amplified by orverlap extension PCR using following primers and KOD FX Neo (TOYOBO) for removing the XhoI site. Primer set 1 is Lp NdeI TilS F, 5’-GGA ATT CCA TAT GAC ACC AAT TCA AAA GTT CAA T-3’ and Lp TilS 805 R, 5’-GCG TCT GCT GCT CTA GCC ACG TCG C-3’. Primer set 2 is Lp TilS 805 F, 5’-GCG ACG TGG CTA GAG CAG CAG ACG C-3’ and Lp XhoI TilS R 5’-CCG CTC GAG ACT CTC GTG TTT TAA GGC TAA AA-3’. Underlines show restriction enzyme sites (NdeI or XhoI). Obtained DNA fragments from primer set 1 and 2 were purified from agarose gel. These two DNA fragments were used as templates for overlap extension PCR using primer set of Lp NdeI TilS F and Lp XhoI TilS R to obtain one fragment with TilS gene with NdeI site at the 5’ end and XhoI site at the 3’ end. The amplified fragment was extracted from the agarose gel and cloned into the NdeI and XhoI sites of the pET22b expression vector. E. coli RosettaTM 2(DE3) was used as the host for the recombinant TilS expression. The C-terminal His-tagged TilS was purified using Ni-SepharoseTM High performance (GE Healthcare). Obtained TilS fraction was concentrated by Amicon Ultra-15 (Millipore). Glycerol was added (final concentration 50%) to the TilS fraction, and the protein was stored at –30 °C. Quantity of the protein was measured with a Bio-Rad protein assay kit (Quick StartTM Bradford Dye Reagent, 1X) using bovine serum albumin as a standard and analysed by 15% SDS-PAGE. Preparation of modified tRNA transcript by TilS The activity of the recombinant TilS was measured at 37 °C as described previously (2). The 50-µl reaction mixture consisted of 100 mM Tris-HCl (pH 7.8), 10 mM KCl, 10 mM MgCl2, 10 mM DTT, 2 mM ATP, 3 µM [14 C(U)]-lysine (11 GBq/mmol, PerkinElmer Life and Analytical Science), 2 µg TilS protein and 0.2 A260 units of tRNA transcript. Synthetic oligo sequences for in vitro tRNAs transcription were described in Supplementary Table SI. We employed two-dimensional TLC for assignment of 14 C-incorporated nucleotide by TilS (15). The 14 C-incorporated tRNA was dissolved in 3 µl of 50 mM ammonium acetate (pH 5.0) and digested with one unit of nuclease P1. A 1-µl aliquot of the sample was spotted onto a thin layer plate (MERCK, TLC Cellulose F; 10 cm x 10 cm) and separated using the solvent systems (a) isobutyric acid, conc. ammonia, water (66: 1: 33, v/v/v), (b) isopropanol, HCl, water (75: 15: 15, v/v/v) and (c) 100 mM sodium phosphate (pH 6.8), ammonium sulphate, 1-propanol (100: 60: 2, v/w/v). Finally, the mass of the modified nucleotide was analysed by MALDI-TOF MS. Results Isolation of L. plantarum tRNAIle species L. plantarum WCFS1 contains five genes that encode isoleucine tRNAs (Transfer RNA Database, http://trna.bioinf.uni-leipzig.de/). One encodes tRNAIle1(GAU), which decodes AUC and AUU codons. Two genes encode tRNAIle2(CAU) (lp_tRNA65, lp_tRNA51), which decodes AUA codons. Although these two genes are annotated as encoding tRNAMet, these anticodons are expected to be modified into LAU by TilS (lp_0545). In addition, there are two genes encoding tRNAIle2(UAU) (lp_tRNA10, lp_tRNA64). The sequences of these two tRNAIle2(UAU) genes differ by only two nucleotides (Fig. 1A). The expression of tRNAIle2(UAU) genes were verified by northern hybridization of total RNAs from L. plantarum and E. coli. A band corresponding to tRNAIle2(UAU) was detected in the L. plantarum total RNA (Supplementary Fig. S1). This result shows that at least one of the tRNAIle2(UAU) genes is expressed. The absence of a band corresponding to tRNAIle2(UAU) in E. coli total RNA confirmed the specificity of the probe used. Sequencing of the RT-PCR products from total RNA from L. plantarum revealed transcripts corresponding to both genes: approximately 75% of the transcripts corresponded to tRNAIle2(UAU)U48, C50 and 25% to tRNAIle2(UAU)C48, U50 (data not shown). We then isolated tRNAIle2(UAU), tRNAIle2(CAU) and tRNAIle1(GAU) from L. plantarum using a solid-phase DNA probe column method (Fig. 1B) (13). The result demonstrates that both tRNAIle2(UAU) and tRNAIle2(CAU) exist as stable species in L. plantarum. Fig. 1 Open in new tabDownload slide Isolation of L. plantarum tRNAIles. (A) Clover leaf structures of L. plantarum tRNAIle2(UAU) and tRNAIle2(CAU), which are products of lp_tRNA10 and lp_tRNA65, respectively. C48 and U50 of tRNAIle2(UAU) are highlighted with numbers. In the other tRNAIle2(UAU) encoded by lp_tRNA64, these positions are U48 and C50. In tRNAIle2(CAU), C34 is modified to L by TilS. Also C20a, U45, U51 and A63, which are substituted by U20a, G45, A51 and U63, respectively, in the other tRNAIle2(CAU) encoded by lp_tRNA51 are highlighted with numbers. (B) Purification of L. plantarum small RNAs and isolation of tRNAIle2(UAU), tRNAIle2(CAU) and tRNAIle1(GAU). Purified RNAs were separated on a 10% polyacrylamide/7 M urea gel and detected by toluidine blue staining. The anticodon of L. plantarum tRNAIle2(CAU) is modified by TilS In most eubacteria, the anticodon of tRNAIle2(CAU) is modified by TilS after transcription. The L modification of the anticodon loop is required for recognition by IleRS and for decoding of AUA codon (16). In the presence of ATP, TilS catalyzes covalent bond formation between the ε-amino group of lysine and C2 of cytosine in the tRNAIle2(CAU) anticodon. The lp_0545 gene of L. plantarum genome is annotated as a tilS gene (11, 17). This gene was cloned into an expression vector, and the recombinant protein was partially purified (Supplementary Fig. S2). TilS activity was measured by the incorporation of [14 C]-lysine into in vitro transcribed tRNAs. In the presence of partially purified recombinant TilS, lysine was incorporated into the tRNAIle2(CAU) transcript, whereas no incorporation was observed for tRNAMet, although this tRNA has the same anticodon sequence as tRNAIle2(CAU) (Fig. 2A). The strict discrimination by TilS between tRNAIle2(CAU) and tRNAMet has been reported for E. coli TilS (16). Alteration of the CAU anticodon of tRNAIle2(CAU) to UAU completely abolished incorporation of lysine by TilS suggesting that the anticodon cytidine is the site of incorporation (Fig. 2A). Fig. 2 Open in new tabDownload slide The C34 of L. plantarum tRNAIle2(CAU) is modified by TilS. (A) Time course of lysidine formation in tRNAIle2(CAU) (circle), tRNAIle2(CAU/UAU) mutant (triangle) and initiator tRNAMet(CAU) (square) catalyzed by TilS. (B) The tRNAIle2(CAU) transcript was incubated with partially purified recombinant TilS, and modified nucleotides were identified by 2 D TLC. Positions of standard markers (pA, pG, pC and pU) are enclosed by dotted circles. (C) Mass spectrum of L. plantarum tRNAIle2(CAU) transcript modified in vitro by purified TilS and digested with RNase A. The spectrum shows the lysidine-34 containing fragment LAUp. Inset spectrum shows the tRNAIle2(CAU) transcript without modification. (D) Detection of lysidine-34 in the native L. plantarum tRNAIle2(LAU) by MALDI-TOF MS. Mass spectrum of the native L. plantarum tRNAIle2(LAU) digested by RNase A shows a lysidine-34 containing fragment LAUp. The spectrum is enlarged around the AACp fragment to show the putative methylation at the position 37. Supplementary Table SII gives theoretical and empirical masses of singly protonated ions derived from the RNase A fragments of the L. plantarum tRNAIle2(LAU). Formation of lysidine was verified first by two-dimensional TLC. The tRNAIle2(CAU) treated with TilS in the presence of [14 C]-lysine was digested with nuclease P1, and the resulting nucleotides were analysed by 2 D TLC (Fig. 2B). The results confirmed the formation of lysidine as shown previously (15). TilS-treated tRNAIle2(CAU) transcript was then digested with pyrimidine-specific RNase A, and samples were analysed by MALDI-TOF MS. Unlike the spectrum of tRNAIle2(CAU) without lysine ligation by TilS (Fig. 2C, inset), a peak at m/z 1087 corresponding to the mass of LAU was observed in the spectrum of tRNAIle2(CAU) treated with TilS (Fig. 2C). Furthermore, the peak of the unique A35U adjacent to the position 34 decreased when L34 was present (data not shown). These results show that modification of cytidine into lysidine prevented cleavage at position 34 by RNase A resulting in the appearance of LAU fragment and a decrease in the amount of the unique AU fragment. From this result, we conclude that C34 is the site of lysidine modification in tRNAIle2(CAU). Finally, we analysed tRNAIle2(CAU) isolated from L. plantarum by MALDI-TOF MS to determine whether lysidine was present. A fragment (m/z 1087) with the mass of LAU was also observed in this sample (Fig. 2D), confirming the lysidine modification in the anticodon in vivo. Hereafter, tRNAIle2(CAU) of L. plantarum will be denoted as tRNAIle2(LAU). We did not detect a fragment at m/z 1126 indicating that the native tRNA does not have N6-threonylcarbamoyladenosine (t6A) at position 37 (a fragment at m/z 1126 with sequence t6AAUp was not detectable); this is in contrast to E. coli tRNAIle(LAU), which does have the t6A modification (18). Instead, the spectrum of L. plantarum tRNAIle2(LAU) shows an ion at m/z 996.27 corresponding to the mass of methylated A37ACp fragment (Fig. 2D, inset). The result suggests the presence of N6-methyladenosine (m6A) at position 37 as is the case for tRNAIle(LAU) from Bacillus subtilis and from Mycoplasma capricolum (19, 20). Characterization of tRNAIle2(UAU) The tRNAIle2(UAU) isolated by solid-phase DNA probe column method was digested with RNase T1 and analysed for post-transcriptional modifications by MALDI-TOF MS. Methylation was detected in the fragment UUCGp. This fragment contains position 54 that is known to be modified into 5-methyluridine in almost all tRNAs (Fig. 3A). No other modifications were observed, even in the anticodon loop, by MALDI-MS (Fig. 3B). In some eukaryotes, AUA codons are decoded by the tRNAIle that has a ΨAΨ anticodon where Ψ stands for pseudouridine (21). Since pseudouridine and uridine have the same molecular mass, these two nucleotides cannot be distinguished by MALDI-MS. In a reverse transcription-based analysis (14), no stops of the reverse transcriptase were observed, indicating that no pseudouridines or other modified nucleotides were present in the anticodon loop of tRNAIle(UAU) (Fig. 4). Fig. 3 Open in new tabDownload slide MALDI mass spectrometry analysis of the native L. plantarum tRNAIle2(UAU). Native L. plantarum tRNAIle2(UAU) was digested with RNase T1 and subjected to MALDI-TOF mass spectrometry. Inserts A and B show regions containing position 54 and the anticodon loop, respectively. Theoretical and measured masses of the resulting fragments are listed in the table. Fig. 4 Open in new tabDownload slide Detection of pseudouridine (Ψ) by CMCT-RT. The regions complementary to primers used for reverse transcription are marked with dashed arrows on the cloverleaves schematics. Primers were specific for tRNAIle2(UAU) or tRNAIle2(LAU). Primer extensions were performed on total RNA from L. plantarum. The total RNAs were treated with CMCT for 2, 10 and 20 min followed or not by alkaline (OH-) treatment (+ or –). Dideoxy DNA sequencing ladders (lanes C, T, A, G) were prepared with the same primers. No pseudouridine in the anticodon of tRNAIle2(UAU) was detected. Of tRNAIle2 variants in L. plantarum, only tRNAIle2(LAU) is charged with isoleucine As both species of tRNAIle2, one with LAU anticodon and another with an unmodified UAU anticodon, exist in L. plantarum, we examined whether both tRNAIle isoforms accept isoleucine. The tRNAIle2(UAU), tRNAIle2(LAU) and tRNAIle1(GAU) isolated from L. plantarum were assayed for aminoacylation with [14 C]-isoleucine in vitro using an L. plantarum S-100 fraction. The tRNAIle1(GAU) was used as a positive control. Charging with isoleucine was observed for tRNAIle2(LAU) and tRNAIle1(GAU) (Fig. 5A). In contrast, tRNAIle2(UAU) was not charged with isoleucine, and the level of radioactivity in this sample was the same as observed without tRNA. Fig. 5 Open in new tabDownload slide Of L. plantarum tRNAIle2 variants, only tRNAIle2(LAU) is charged with isoleucine. (A) Ile charging of native tRNAIle2(LAU) and tRNAIle2(UAU) purified from L. plantarum. Native tRNAIle1(GAU) was used as a positive control. Ile acceptance activities were measured by [14 C]Ile incorporation in the presence of L. plantarum S-100. Error bars are SEM for three independent experiments. (B) Ile charging activities of L. plantarum tRNAIle2 transcript and its anticodon variants. Specificity of isoleucine charging was further tested using in vitro transcribed tRNAIle2(LAU), tRNAIle2(CAU) and a mutant of this tRNA with an UAU anticodon as substrates. tRNA substrates were prepared in vitro as described above. Only tRNAIle2(LAU) was charged with isoleucine (Fig. 5B). The result agrees with a previous report that showed lysidine at wobble position of the anticodon of tRNAIle2 is a critical determinant for charging with isoleucine (1). Less isoleucine was incorporated into the in vitro prepared tRNAIle2(LAU) (approximately 50 pmol/A260 in 10 min) compared to the tRNAIle2(LAU) isolated from cells (Fig. 5A and B). This is likely to be due to the incomplete lysidylation of the transcript (estimated to be 50%) and possible contributions of other modifications in tRNAIle2(LAU), such as the previously described methylation at position 37, to the charging efficiency (22). Discussion AUA is a rarely used codon in most organisms. For example, in E. coli, AUA accounts for less than 0.5% of the 1,356,539 codons used in annotated genes. Our analysis showed that in L. plantarum, AUA codons are used even less frequently than this. Of the 920,850 codons in annotated genes, the AUA codon accounts for less than 0.3%. Within the three isoleucine codons (AUA, AUC, AUU), AUA codons represent less than 5% and about 60% of L. plantarum genes do not contain a single AUA codon (Supplementary Fig. S3). In bacteria, a strong correlation is observed between the frequency of codon usage and the concentration of the tRNA decoding the codon (23). Further, the concentrations of individual tRNAs are proportional to the copy numbers of the respective genes in E. coli and B. subtilis (24–26). Thus, the number of genes coding for tRNAIle2(CAU) and tRNAIle2(UAU) in L. plantarum is disproportionate given how rare the AUA codons are. Here, we showed that two tRNA species, tRNAIle2(CAU) and tRNAIle2(UAU), capable of decoding AUA codons are expressed in L. plantarum. As observed in many other bacterial species, we found that tRNAIle2 with the CAU anticodon is modified into LAU and charged with isoleucine to fulfil its role in translation. The tRNAIle2 with the UAU anticodon was not charged with isoleucine. Sequence elements that are important for IleRS have been identified (16, 22) (Supplementary Fig. S4). L. plantarum tRNAIle2(LAU) possesses all the known IleRS determinants, whereas tRNAsIle2(UAU) is missing some, likely explaining why the former is charged with isoleucine and the latter is not. In the absence of lysidine modification, tRNAIle2(LAU) possesses most positive determinants for recognition by MetRS (27) (Supplementary Fig. S4). Lysidine is a critical negative determinant for MetRS in E. coli (1, 16). Here, the modification of the anticodon of tRNAIle2(LAU) by TilS also plays a crucial role in determining the tRNA identity also in L. plantarum. From the crystal structure of Staphylococcus aureus IleRS in complex with E. coli tRNAIle1 (28) and amino acid sequence comparison of IleRSs from various species, Suzuki and co-workers proposed how different IleRSs recognize LAU or UAU anticodons (3). They reported that Trp890 (numbering for S. aureus IleRS) in the C-terminal Zn-binding domain of IleRS is well conserved in species bearing a tilS homologue. In bacterial species not containing a tilS homologue but having tRNAIle2(UAU), the Trp890 is replaced with arginine (M. mobile), lysine (Mycoplasma haemofelis), or leucine (Mycoplasma suis). Mutation of the tryptophan in E. coli IleRS to arginine results in a 10-fold increase in recognition of tRNAIle2(UAU), while the activity toward tRNAIle2(LAU) is maintained (3). Interestingly, even though IleRS of L. plantarum bears arginine at the corresponding position (Supplementary Fig. S5), the enzyme did not charge the tRNAIle2(CAU/UAU) mutant nor the tRNAIle2(UAU). This suggests that L. plantarum IleRS distinguishes the LAU anticodon from the UAU anticodon by a mechanism that differs from that of the E. coli enzyme. Based on the study of AUA decoding system in M. mobile, the evolution of AUA decoding system has been proposed (3). According to this theory, majority of bacteria now existing are at the stage of evolution where tRNAIle with CAU anticodon is modified into LAU by TilS and the anticodon is recognized specifically by IleRS but not by MetRS. This system ensures fidelity of decoding. From this state, in some bacteria, the tRNAIle(TAT) gene would have been generated by the duplication of the tRNAIle(CAT) gene followed by a C-to-T transition at the wobble position of one gene. In such bacteria, IleRS might have evolved to recognize the UAU anticodon. Once IleRS acquired sufficient recognition of UAU anticodon, some bacteria might have lost tilS as has been observed for M. mobile. In this bacterium, evolution has made ribosomes to be capable of preventing misreading of AUG codon by the UAU anticodon. For L. plantarum the situation seems to be outside of this schema as the sequence of tRNAIle(UAU) differs significantly from that of tRNAIle(LAU) in terms of IleRS recognition determinants and the presence of long variable loop. This may also explain, in part, why tRNAIle(UAU) is not charged with isoleucine and may suggest that this tRNA does not function for canonical AUA decoding. Interestingly, both tRNAIle(TAT) genes are found in uninducible prophage sequences of L. plantarum WCFS1 genome (29) suggesting that these tRNAs derived from phages. Prophages contribute to increase the competitiveness of the bacterial populations in their ecological niches (30). A bioinformatic analysis of metagenomic data revealed that stop-to-sense codon reassignment is extensively found in prophages (31). In the case of phages found in the human oral cavity environments, a noncanonical tRNA in the phage is suspected to be responsible for this codon reassignment. Likewise tRNAIle2(UAU) might be involved in mechanisms that leads to genetic code flexibility (32) that confers L. plantarum high survival ability for various environmental changes. Besides the canonical role in translation, tRNAs and tRNA-like molecules have diverse functions. These include enzymatic modification of proteins leading to signalling for degradation of the labelled proteins, cell envelope modification and synthesis of small molecules such as antibiotics (33). tRNAs can also function as sensor-regulator molecules as in the case of T-box riboswitches (34) or in the stringent response (35). Most of these functions, together with canonical translation, utilize the tRNA’s ability to be charged with amino acids. As mentioned above, we did not observe charging of isoleucine to tRNAIle2(UAU). One of the peculiar feature of tRNAIle2(UAU) is the unique secondary structure with a long variable loop. This is characteristic of class II tRNAs, including leucine and serine tRNAs. We tested charging of tRNAIle2(UAU) with these amino acids, but we did not observe incorporation of these amino acids (data not shown). However, it should be noted that L. plantarum is capable to inhabit diverse environmental niches, therefore it may be possible that charging of tRNAIle2(UAU) occurs in certain conditions and/or the tRNA functions to respond or sense environmental changes. It is also possible that tRNAIle2(UAU) is charged with amino acid other than isoleucine. These possibilities should be clarified. To conclude we showed that tRNAIle2(LAU) and tRNAIle2(UAU) are expressed in L. plantarum creating redundancy in tRNAs that would decode rare AUA codons. However, we found that tRNAIle2 (UAU) is not charged with isoleucine. This contrasts with efficient charging of tRNAIle2 (LAU) with isoleucine to fulfil its role in translation. Altogether, the results suggest that tRNAIle2 (LAU) functions as canonical tRNA, while tRNAIle2 (UAU) might display distinct functions in the cell. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements The authors thank Dr. Ro Osawa (Kobe University) for the gift of L. plantarum WCFS1 strain; and Dr. Takeo Suzuki (the University of Tokyo) and Dr. Ikuko Masuda-Nishimura (Kikkoman Corp.) for their technical suggestions. The authors also thank Dr. Henri Grosjean for useful discussions. Funding This work was supported by Kurata Grants from the Kurata Memorial Hitachi Science and Technology Foundation (to C.T.), by a grant for young researchers from JGC-Scholarship Foundation (to C.T.) by Grants in Aid for Scientific Research from the Japan Society for Promotion of Science (16K18493 to C.T.) and by grants from CNRS PRC 23556 (to S.Y.) and Sumitomo Foundation (101071, 111344 to S.Y.). Funding for open access charges is provided by CNRS funding to S.Y. 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USA 70 , 1564 – 1568 Google Scholar Crossref Search ADS WorldCat Abbreviations Abbreviations CMCT 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate HPA hydroxypicolinic acid IleRS isoleucyl-tRNA synthetase L lysidine m6A N6-methyladenosine MALDI-TOF MS matrix assisted laser desorption ionisation - Time of flight mass spectrometry MetRS methionyl-tRNA synthetase Ψ pseudouridine R purine RT reverse transcription SEM standard error of the mean t6A N6-threonylcarbamoyladenosine TilS tRNAIle-lysidine synthetase TLC thin-layer chromatography © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society.

Journal

The Journal of Biochemistry – Oxford University Press

Published: Mar 1, 2018

Keywords: anticodon; isoleucine; transfer rna; lactobacillus

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