what resemblance does the nucleotide sequence of trna anticodons bear to the original dna sequence
Abstract
In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (Fifty) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through postal service-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for instance, 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 specially in the anticodon sequence. Nosotros found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is non. Our results propose that the tRNAIle2 back-up may exist related to unlike roles of these tRNAs in the jail cell.
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 K) with identical nucleotides at the get-go and 2nd positions code for the same amino acids (due east.1000. CUA and CUG code for leucine). This necessitates strict discrimination of the codons for the tRNAs decoding AUA and AUG codons. Leaner employ two different strategies to cope with this problem.
In virtually all bacteria, the AUA codon is translated past 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 machinery of the bigotry of AUR codons by the modification has been characterized in Escherichia coli. The significance of the lysidine modification was first demonstrated past 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 but specifies the codon-anticodon interaction only likewise affects the tRNA identity. The protein responsible for the lysidine germination 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 2nd strategy is used by bacteria that lack tilS homologues and possess tRNAIle genes with an UAU anticodon (3, four). Mycoplasma mobile is an example of such species, and the decoding organization for AUA codon has been studied recently (3). The UAU anticodon of tRNAIle2 of M. mobile is unmodified, and information technology has been suggested that ribosomes from M. mobile have acquired the chapters to prevent misreading of the AUG codon by the UAU anticodon. Grand. mobile is a Mollicutes, a parasitic bacterium. Like other Mollicutes, Thousand. mobile has an extremely minor genome of 770 kilobases (v). Mollicutes abound autonomously by maintaining a minimum fix of necessary genes (6). The decoding organisation used for AUA codon by M. mobile enabled loss of tilS gene.
We speculate that a third strategy might be, used by Lactobacillus plantarum for example, in which genes coding for a tilS homologue and tRNAIle with a CAU anticodon be in addition to the gene that encodes tRNAIle with the UAU anticodon. Fifty. plantarum is a Gram-positive bacterium that is ordinarily found in starting cultures for food fermentation (7, viii). This bacterium possesses loftier survival chapters in the homo gastrointestinal tract (9) and is likely to have propitious effects on human health (10). The genome of 50. plantarum is i of the largest known amidst lactic acid-metabolizing bacteria (xi, 12). This large genome may enable this bacterium to inhabit diverse ecology niches by utilizing a wide range of carbohydrates (12). It is not clear, however, why the bacterium would possess a redundant AUA decoding system. In this study, we used 50. plantarum as a model organism to exam for the potential coexistence of isoacceptor tRNAs bearing dissimilar anticodons that would decode the AUA codon. We establish that both tRNAs are indeed expressed in L. plantarum. As expected, the C of the CAU anticodon of tRNAIle2(CAU) is modified to 50 by the tilS homologue. We found this tRNA to be charged with isoleucine for use in translation. On the opposite, tRNAIle2(UAU) lacks tRNA modifications peculiarly 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 civilisation
The civilisation 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 goop) agar (containing i.5% agar, w/v) or cultured statically in MRS broth at 37 °C.
RNA preparation
50. plantarum total RNA was extracted using ISOGEN II (Japan Gene) co-ordinate to manufacturer's manual with slight modifications. Frozen L. plantarum cells (ii g) were basis in a chilled mortar for five–x min. ISOGEN Ii solution (4 ml) was added to the mortar, and cells were further ground for ten–20 min. The cell pause was transferred to a tube, and then treated according to the ISOGEN 2 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 (xiii). The three'-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 x% polyacrylamide/vii G urea gel.
Northern hybridization
The northern hybridization was performed every bit reported previously (thirteen). Total RNAs (0.2 A260 units) from L. plantarum and E. coli were separated on a 10% polyacrylamide/7 Thousand urea gel, and and so the gel was blotted onto Hybond Due north+ (GE healthcare). The hybridization was performed at 50 °C for over 12 h. Sequences of the constructed 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 analysis was performed using the S-100 fraction prepared from L. plantarum and [fourteen C(U)]-Ile (11.03 GBq/mmol) from Moravek Biochemicals, Inc. Amino acrid 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 past PCR using synthetic DNA oligonucleotides ( Supplementary Table SI).
Mass spectrometry analyses
After purification using the solid-phase Deoxyribonucleic acid 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 xxx min at 37 °C in 5 μl of 20 mM 3-hydroxypicolinic acid (HPA) with thirty units of RNase T1 (Roche) or 5 μl of 100 mM ammonium acetate (pH vii.0) containing 2 µg of RNase A (Roche). The residual cyclic 2', iii'-phosphates formed during RNase T1 digestion were removed by add-on of HCl to a last concentration of 0.one M and incubation at room temperature for 5 min. A i μl aliquot of the digest was mixed with 9 µl HPA (40 mg/ml in water: acetonitrile 50 : 50), and ane µl of the mixture was spotted on the MALDI plate and air-stale ('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 one-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). Earlier using for reverse transcription, the total RNA was denatured at 94 °C for 3 min, and and so chilled on ice immediately. For detecting nucleotide at position 34, a primer that is complementary to the variable region at the T-loop of 50. plantarum tRNAIle2(UAU) was used ( Supplementary Table SI). Every bit 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). Deoxyribonucleic acid sequencing with an fmol DNA Cycle Sequencing Organisation (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 cistron in L. plantarum genome has been annotated equally tilS (NCBI: http://world wide web.ncbi.nlm.nih.gov). Since one restriction enzyme (XhoI) site exists within the factor, DNA fragment of the lp_0545 gene was amplified past orverlap extension PCR using following primers and KOD FX Neo (TOYOBO) for removing the XhoI site. Primer set 1 is Lp NdeI TilS F, v'-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 prepare two is Lp TilS 805 F, 5'-GCG ACG TGG CTA GAG CAG CAG ACG C-3' and Lp XhoI TilS R five'-CCG CTC GAG Deed CTC GTG TTT TAA GGC TAA AA-three'. Underlines show restriction enzyme sites (NdeI or XhoI). Obtained DNA fragments from primer set 1 and ii were purified from agarose gel. These two Deoxyribonucleic acid fragments were used as templates for overlap extension PCR using primer prepare 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' finish. The amplified fragment was extracted from the agarose gel and cloned into the NdeI and XhoI sites of the pET22b expression vector. Due east. coli RosettaTM 2(DE3) was used as the host for the recombinant TilS expression. The C-concluding His-tagged TilS was purified using Ni-SepharoseTM High performance (GE Healthcare). Obtained TilS fraction was full-bodied past Amicon Ultra-15 (Millipore). Glycerol was added (last concentration 50%) to the TilS fraction, and the protein was stored at –xxx °C. Quantity of the poly peptide 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 seven.8), 10 mM KCl, 10 mM MgClii, 10 mM DTT, 2 mM ATP, 3 µM [xiv C(U)]-lysine (11 GBq/mmol, PerkinElmer Life and Belittling Science), two µg TilS protein and 0.2 A260 units of tRNA transcript. Synthetic oligo sequences for in vitro tRNAs transcription were described in Supplementary Tabular array SI. Nosotros employed ii-dimensional TLC for assignment of 14 C-incorporated nucleotide by TilS (15). The 14 C-incorporated tRNA was dissolved in iii µl of fifty mM ammonium acetate (pH 5.0) and digested with i unit of nuclease P1. A ane-µl aliquot of the sample was spotted onto a thin layer plate (MERCK, TLC Cellulose F; ten cm x ten cm) and separated using the solvent systems (a) isobutyric acrid, conc. ammonia, water (66: one: 33, v/v/5), (b) isopropanol, HCl, water (75: 15: 15, five/v/5) and (c) 100 mM sodium phosphate (pH vi.8), ammonium sulphate, i-propanol (100: 60: 2, 5/w/5). Finally, the mass of the modified nucleotide was analysed by MALDI-TOF MS.
Results
Isolation of L. plantarum tRNAIle species
L. plantarum WCFS1 contains 5 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, in that location are two genes encoding tRNAIle2(UAU) (lp_tRNA10, lp_tRNA64). The sequences of these two tRNAIle2(UAU) genes differ past but ii nucleotides (Fig. 1A). The expression of tRNAIle2(UAU) genes were verified by northern hybridization of total RNAs from L. plantarum and E. coli. A ring corresponding to tRNAIle2(UAU) was detected in the L. plantarum full RNA ( Supplementary Fig. S1). This result shows that at least ane of the tRNAIle2(UAU) genes is expressed. The absence of a ring 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 50. plantarum revealed transcripts respective 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 and so isolated tRNAIle2(UAU), tRNAIle2(CAU) and tRNAIle1(GAU) from L. plantarum using a solid-stage Deoxyribonucleic acid probe column method (Fig. 1B) (13). The result demonstrates that both tRNAIle2(UAU) and tRNAIle2(CAU) exist as stable species in 50. plantarum.
Fig. i
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 past lp_tRNA64, these positions are U48 and C50. In tRNAIle2(CAU), C34 is modified to 50 past 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 past toluidine blue staining.
Fig. 1
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 past 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 past 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 ten% polyacrylamide/7 M urea gel and detected by toluidine blue staining.
The anticodon of 50. plantarum tRNAIle2(CAU) is modified by TilS
In about 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 (xvi). 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 equally a tilS gene (xi, 17). This gene was cloned into an expression vector, and the recombinant poly peptide was partially purified ( Supplementary Fig. S2). TilS activity was measured by the incorporation of [fourteen 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 past 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 past TilS suggesting that the anticodon cytidine is the site of incorporation (Fig. 2A).
Fig. 2
The C34 of L. plantarum tRNAIle2(CAU) is modified by TilS. (A) Time course of lysidine germination 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 past 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 Tabular array SII gives theoretical and empirical masses of singly protonated ions derived from the RNase A fragments of the 50. plantarum tRNAIle2(LAU).
Fig. 2
The C34 of L. plantarum tRNAIle2(CAU) is modified by TilS. (A) Time grade of lysidine germination 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 past ii D TLC. Positions of standard markers (pA, pG, pC and pU) are enclosed past dotted circles. (C) Mass spectrum of L. plantarum tRNAIle2(CAU) transcript modified in vitro past 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) past MALDI-TOF MS. Mass spectrum of the native L. plantarum tRNAIle2(LAU) digested past 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 50. plantarum tRNAIle2(LAU).
Formation of lysidine was verified first by two-dimensional TLC. The tRNAIle2(CAU) treated with TilS in the presence of [xiv C]-lysine was digested with nuclease P1, and the resulting nucleotides were analysed by two 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 past MALDI-TOF MS. Unlike the spectrum of tRNAIle2(CAU) without lysine ligation by TilS (Fig. 2C, inset), a peak at g/z 1087 corresponding to the mass of LAU was observed in the spectrum of tRNAIle2(CAU) treated with TilS (Fig. 2C). Furthermore, the summit of the unique A35U next to the position 34 decreased when L34 was present (information not shown). These results testify 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 outcome, nosotros 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 make up one's mind whether lysidine was nowadays. A fragment (1000/z 1087) with the mass of LAU was also observed in this sample (Fig. 2D), confirming the lysidine modification in the anticodon in vivo. Time to come, 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 North 6-threonylcarbamoyladenosine (tviA) at position 37 (a fragment at m/z 1126 with sequence thalf-dozenAAUp was not detectable); this is in dissimilarity to Due east. coli tRNAIle(LAU), which does have the t6A modification (xviii). Instead, the spectrum of Fifty. plantarum tRNAIle2(LAU) shows an ion at m/z 996.27 corresponding to the mass of methylated A37ACp fragment (Fig. 2D, inset). The outcome suggests the presence of Due north 6-methyladenosine (grand6A) at position 37 as is the case for tRNAIle(LAU) from Bacillus subtilis and from Mycoplasma capricolum (19, 20).
Label 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 five-methyluridine in near 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 aforementioned molecular mass, these two nucleotides cannot exist distinguished by MALDI-MS. In a reverse transcription-based analysis (14), no stops of the opposite transcriptase were observed, indicating that no pseudouridines or other modified nucleotides were present in the anticodon loop of tRNAIle(UAU) (Fig. iv).
Fig. three
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. iii
MALDI mass spectrometry analysis of the native L. plantarum tRNAIle2(UAU). Native 50. 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
Detection of pseudouridine (Ψ) past 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 past alkaline (OH-) treatment (+ or –). Dideoxy Deoxyribonucleic acid sequencing ladders (lanes C, T, A, One thousand) were prepared with the same primers. No pseudouridine in the anticodon of tRNAIle2(UAU) was detected.
Fig. iv
Detection of pseudouridine (Ψ) past CMCT-RT. The regions complementary to primers used for opposite 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 Fifty. plantarum. The full RNAs were treated with CMCT for 2, ten and 20 min followed or not past alkali metal (OH-) handling (+ or –). Dideoxy Deoxyribonucleic acid sequencing ladders (lanes C, T, A, M) were prepared with the same primers. No pseudouridine in the anticodon of tRNAIle2(UAU) was detected.
Of tRNAIle2 variants in L. plantarum, but 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, nosotros examined whether both tRNAIle isoforms accept isoleucine. The tRNAIle2(UAU), tRNAIle2(LAU) and tRNAIle1(GAU) isolated from L. plantarum were assayed for aminoacylation with [xiv C]-isoleucine in vitro using an L. plantarum S-100 fraction. The tRNAIle1(GAU) was used every bit a positive command. 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 equally observed without tRNA.
Fig. 5
![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.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jb/163/3/10.1093_jb_mvx075/1/m_mvx075f5.jpeg?Expires=1651136852&Signature=17oi0nQPplQhbDTR~56aDLPYXfHJGIbFSX11SAYXtRoozfcKnZx01eYW6nSg1mf78uu0KFLO-ltcxMwdPm7EULZCFuZMf931sz3nRqIxD-eu-98nrU0oyTGZxdId3AeYUF82JBoxbkwwxHm0orWGMxxn9QflO9Luz-w4tPEDzCFJT4B0OJEpmCVOEP04-rVi-jog6Cj2INPoR6ByFJ8JyNJJNpx-tN4RgyHYxDjAgDJpIKtcXE8~uEuiN-9C5ehyFyCfCFmzZhGLTFGvAIEllyR-zE6fd1V-YvqowikqOmEUfzvwpPGaQq6bO-T0ciRbggjh7BpH9qSqt-~diG379g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Of Fifty. 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 equally a positive command. Ile acceptance activities were measured past [14 C]Ile incorporation in the presence of Fifty. plantarum Due south-100. Error bars are SEM for three contained experiments. (B) Ile charging activities of L. plantarum tRNAIle2 transcript and its anticodon variants.
Fig. 5
Of L. plantarum tRNAIle2 variants, simply 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 [fourteen C]Ile incorporation in the presence of 50. plantarum S-100. Fault confined 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 in a higher place. Only tRNAIle2(LAU) was charged with isoleucine (Fig. 5B). The event agrees with a previous written report that showed lysidine at wobble position of the anticodon of tRNAIle2 is a disquisitional determinant for charging with isoleucine (one). 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 exist due to the incomplete lysidylation of the transcript (estimated to exist 50%) and possible contributions of other modifications in tRNAIle2(LAU), such every bit 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 Due east. 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%. Inside the 3 isoleucine codons (AUA, AUC, AUU), AUA codons represent less than five% and near 60% of L. plantarum genes do non contain a single AUA codon ( Supplementary Fig. S3). In leaner, a strong correlation is observed between the frequency of codon usage and the concentration of the tRNA decoding the codon (23). Farther, the concentrations of individual tRNAs are proportional to the copy numbers of the respective genes in Due east. coli and B. subtilis (24–26). Thus, the number of genes coding for tRNAIle2(CAU) and tRNAIle2(UAU) in Fifty. plantarum is disproportionate given how rare the AUA codons are.
Here, we showed that 2 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). Fifty. plantarum tRNAIle2(LAU) possesses all the known IleRS determinants, whereas tRNAsIle2(UAU) is missing some, probable explaining why the old is charged with isoleucine and the latter is non. In the absence of lysidine modification, tRNAIle2(LAU) possesses nigh positive determinants for recognition by MetRS (27) ( Supplementary Fig. S4). Lysidine is a critical negative determinant for MetRS in E. coli (ane, 16). Here, the modification of the anticodon of tRNAIle2(LAU) by TilS as well plays a crucial role in determining the tRNA identity besides in Fifty. plantarum.
From the crystal structure of Staphylococcus aureus IleRS in complex with East. 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-final 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 ten-fold increase in recognition of tRNAIle2(UAU), while the activeness toward tRNAIle2(LAU) is maintained (three). 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 past 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, bulk of bacteria now existing are at the stage of development where tRNAIle with CAU anticodon is modified into LAU by TilS and the anticodon is recognized specifically past IleRS but not past MetRS. This system ensures allegiance of decoding. From this state, in some bacteria, the tRNAIle(TAT) gene would take 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 accept evolved to recognize the UAU anticodon. In one case IleRS caused sufficient recognition of UAU anticodon, some leaner might have lost tilS as has been observed for M. mobile. In this bacterium, evolution has fabricated ribosomes to be capable of preventing misreading of AUG codon past the UAU anticodon. For L. plantarum the state of affairs seems to exist outside of this schema equally 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 role 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 assay of metagenomic data revealed that cease-to-sense codon reassignment is extensively institute in prophages (31). In the instance of phages constitute 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 Fifty. plantarum high survival ability for various environmental changes.
Too the approved function in translation, tRNAs and tRNA-similar molecules take 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 tin too function equally sensor-regulator molecules as in the instance of T-box riboswitches (34) or in the stringent response (35). Nigh of these functions, together with canonical translation, use the tRNA's ability to be charged with amino acids. As mentioned to a higher place, we did not detect charging of isoleucine to tRNAIle2(UAU). One of the peculiar characteristic 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 nosotros did not notice incorporation of these amino acids (data not shown). However, it should exist noted that 50. plantarum is capable to inhabit diverse environmental niches, therefore it may exist possible that charging of tRNAIle2(UAU) occurs in certain conditions and/or the tRNA functions to respond or sense ecology changes. It is as well possible that tRNAIle2(UAU) is charged with amino acid other than isoleucine. These possibilities should be antiseptic.
To conclude nosotros showed that tRNAIle2(LAU) and tRNAIle2(UAU) are expressed in 50. plantarum creating redundancy in tRNAs that would decode rare AUA codons. Even so, we plant 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 propose that tRNAIle2 (LAU) functions as canonical tRNA, while tRNAIle2 (UAU) might brandish distinct functions in the jail cell.
Supplementary Data
Supplementary Data are bachelor 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 Academy of Tokyo) and Dr. Ikuko Masuda-Nishimura (Kikkoman Corp.) for their technical suggestions. The authors too thank Dr. Henri Grosjean for useful discussions.
Funding
This piece of 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.) past Grants in Aid for Scientific Inquiry from the Japan Society for Promotion of Science (16K18493 to C.T.) and by grants from CNRS PRC 23556 (to Southward.Y.) and Sumitomo Foundation (101071, 111344 to Due south.Y.). Funding for open up admission charges is provided by CNRS funding to S.Y.
Conflict of Interest
None declared.
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Abbreviations
-
CMCT
1-cyclohexyl-3-(ii-morpholinoethyl) carbodiimide metho-p-toluene sulfonate
-
HPA
-
IleRS
isoleucyl-tRNA synthetase
-
L
-
m6A
-
MALDI-TOF MS
matrix assisted laser desorption ionisation - Fourth dimension of flight mass spectrometry
-
MetRS
methionyl-tRNA synthetase
-
Ψ
-
R
-
RT
-
SEM
standard error of the hateful
-
t6A
N 6-threonylcarbamoyladenosine
-
TilS
tRNAIle-lysidine synthetase
-
TLC
thin-layer chromatography
Abbreviations
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