Nucleic Acids Research

Plant materials

Four different dicotyledonous plant species were used in this study: Arabidopsis thaliana ecotype Wassilewskija; lupin (Lupinus luteus var. sulfa); tobacco (Nicotiana tabacum Petit Havana line SRI); pea (Pisum sativum L.).

DNA and RNA isolation

DNAs from young seedlings of Arabidopsis and lupin, leaves of tobacco and embryonic axes of pea were isolated as described (12). A tRNA-enriched RNA fraction (hereafter referred to as tRNAs) from Arabidopsis and lupin plants and transformed Arabidopsis calli was prepared by a modified single-step extraction method (13). Guanidinium thiocyanate (8 M) in the extraction buffer was replaced with guanidine hydrochloride (4 M). In this buffer >90% of the RNA isolated was found to be total tRNAs by gel analysis. To isolate tRNAs for acid gel electrophoresis the extraction buffer was adjusted to pH 5.0 to prevent hydrolysis of aminoacyl bonds.

PCR amplification and sequencing of plant tRNA^His genes and cDNAs

PCR amplification of nuclear tRNA^His genes from the four different plant DNAs were performed with primers His5[prime] and His3[prime] (Fig. 2), corresponding to the 5[prime]-distal and complementary to the 3[prime]-distal region of an Arabidopsis nuclear tRNA^His gene (7). The conditions employed followed those of Akama et al. (14). The DNA samples were separated on an 8% polyacrylamide gel, followed by elution of the DNA from the gel slices in TE/0.1% SDS buffer overnight. The products were phosphorylated at the 5[prime]-ends with T4 polynucleotide kinase and then ligated into the EcoRV site of pBluescriptII KS+ (Stratagene). For synthesis of single-stranded cDNAs of cytosolic tRNAs^His from Arabidopsis and lupin tRNA mixtures the tRNAs (0.5 µg) were incubated in the presence of 170 U DNase I for 30 min and then denatured at 80°C for 10 min. Reverse transcription was carried out in 100 mM Tris-HCl, pH 8.2, 10 mM MgCl₂, 140 mM KCl, 0.5 mM each of the four dNTPs, 10 U RNase inhibitor (Life Technologies) using 200 U AMV reverse transcriptase with the His3[prime] primer (20 pmol) at 42°C for 1 h. Double-stranded cDNAs were generated by PCR amplification of the single-stranded cDNAs with primers His5[prime] and His3[prime]. They were cloned as described above. DNA sequencing was performed using a semi-automated DNA sequencer (model 373A; Applied Biosystem).

Construction of mutated tRNA^His genes

Mutations were introduced into the Arabidopsis gene coding for tRNA^His by in vitro site-directed mutagenesis as described (15). A 175 bp RsaI-AluI sub-fragment carrying an Arabidopsis tRNA^His gene, originally derived from clone t-19 (7), was inserted into the EcoRV site of pBluescriptII KS+ to make tHis/wt. Two different oligonucleotide primers, C34A36 and U54, shown in Figure 2, were used to construct two different mutated versions of this plasmid, i.e. tHis/amber (anticodon changed to one complementary to the amber codon) and tHisU54 (substitution of T₅₄ for C₅₄). Introduction of these mutations into the correct positions was confirmed by sequencing.

Plant transformation

To construct a Ti plasmid including an Arabidopsis tRNA^His gene the binary vector pBI121 (Clonetech) was first cleaved by BamHI and EcoRI to remove the [beta]-glucuronidase gene-nopaline synthetase terminator cassette and then filled-in and self-ligated, resulting in pBI121[Delta]GUS. HindIII-XbaI sub-fragments of tHis/wt or tHis/amber were then inserted between the HindIII and XbaI sites of pBI121[Delta]GUS. These plasmids (pBItHis/wt and pBItHis/amber) were used to transform Agrobacterium tumefaciens strain GV3101 (16) by electroporation (17). Infection of Arabidopsis plant tissues with agrobacteria, co-cultivation and selection of transformed calli were performed by the hypocotyl transformation method as described (18). For RNA isolation ~10 independent transformed calli were pooled before extraction.

RNA blot hybridization

Plant tRNAs were separated by 10% polyacrylamide/8 M urea gel electrophoresis. The tRNAs were capillary blotted onto Hybond N+ membrane (Amersham). Two different kinds of DNA probes were used for hybridization. The entire Arabidopsis tRNA^His coding region was PCR-amplified with primers His5[prime] and His3[prime] in the presence of DIG-11-dUTP. Oligonucleotides C54, U54, Atsp, Llsp, His3[prime] and C34A36 (Fig. 2) were all 3[prime]-end labelled with DIG-11-ddUTP by terminal deoxynucleotidyl transferase. Hybridization was carried out overnight in 6× SSC (1× SSC = 150 mM NaCl, 15 mM sodium citrate), 0.1% N-lauroylsarcosine, 0.02% SDS, 1% (w/v) blocking reagent at 45°C for every oligoprobe. In the case of the PCR-amplified probe the buffer was the same except that formamide was added to a final concentration of 50% (v/v). After hybridization, washing was first carried out in 6× SSC, 0.1% SDS at room temperature and then twice successively at 45°C for probes C54 and U54 and at 50°C for probes Atsp and Llsp. The PCR probe was washed in 0.1× SSC at 65-68°C. Immunochemical detection of probes hybridized with tRNA followed the manufacturer's protocol (Boehringer). To detect transcripts of tHis/amber and tHis/wt in transformed cells probe C34A36 was used together with His3[prime] as a control. Detailed hybridization conditions of this experiment are described in the legend to Figure 7.

In vitro transcription of Arabidopsis tRNA^His genes in tobacco nuclear extract

Extraction of crude nuclear proteins from tobacco BY-2 cells and the in vitro transcription assay were essentially as described (19). Plasmid DNAs were purified by CsCl/EtBr ultracentrifugation or using a Qiagen plasmid kit (Qiagen) and their concentrations were measured with PicoGreen double-stranded DNA quantitation reagent (Molecular Probes). For the transcription assay 0.1 pmol template plasmid DNA was added to the reaction cocktail [20 mM HEPES-KOH, pH 7.9, 10% (v/v) glycerol, 3 mM MgSO₄, 80 mM KOAc, 0.1 mM EGTA, 2 mM DTT, 15 U pancreatic RNase inhibitor (Takara, Japan), 1 µg/ml [alpha]-amanitin] containing 30 µg nuclear extract which was then placed on ice for 10 min. Substrate NTPs (0.5 mM each ATP, CTP and GTP, 40 µM UTP and 92.5 kBq [[alpha]-³²P]UTP) were added to the reaction mixtures to start the reactions and then transcription was allowed to proceed at 28°C for 2 h. The resulting transcripts were fractionated on a 12% acrylamide/8 M urea gel. The gel was analysed using a Bio-Imaging Analyzer BAS-2000 II (Fuji Photo Film, Japan).

Detection of in vivo aminoacylation of plant tRNA^His

Acid gel electrophoresis (20) was used to detect the level of charged and uncharged tRNA^His in cells. tRNAs were fractionated on a 12% polyacrylamide gel in 8 M urea/0.1 M sodium acetate buffer, pH 5.0, at 100 V (~7 V/cm) in a cold room for 24 h. As a control, deacylated tRNA samples (incubated in 50 mM Tris-HCl, pH 8.0, 2.5 mM EDTA at 37°C for 1 h) were loaded in parallel. Transfer of the RNA to a membrane, preparation of probes (C54 and U54; Fig. 2) and conditions of hybridization, washing and detection were as described above in RNA hybridization.

RESULTS

Plant tRNA^His genes contain a cytidine at position 54

The nucleotide sequence of an Arabidopsis tRNA^His gene contains C₅₄ (7), whereas lupin cytosolic tRNA^His has been reported to contain U₅₄ (8). To investigate the nature of this nucleotide in other Arabidopsis tRNA^His genes and in other plant species PCR analysis was carried out on four different plant DNAs with primers based on the Arabidopsis and lupin coding sequences (Fig. 2). PCR products (72 nt) were cloned and sequenced. As summarized in Figure 3, they all code for cytosolic tRNA^His as judged by their potential cloverleaf structure, their similarity to the original Arabidopsis gene and the presence of a histidine anticodon triplet (GTG). Although several substitutions were detected at positions 20A (D loop), 25 (D stem), 26 (between the D stem and anticodon stem) and 44 (variable loop), none of these would be expected to influence tRNA structure or gene expression. On the other hand, position 54 was always a C in all plant clones analysed. In parallel, the amplified products were tested by restriction enzyme analysis. The original Arabidopsis gene contains CTCGAA (nt 54-59), whereas the presence of T₅₄ would result in a target site (TTCGAA) for NspV, thus allowing us to determine the nucleotide (C or T) at postion 54. Figure 4 shows that PCR products from all plant species tested lack an NspV recognition site, strongly suggesting that all the amplified tRNA^His genes contain C₅₄. From the sequence heterogeneity in the Arabidopsis PCR clones we can tell that these sequences derive from at least four different genes. Taken together with an estimation of ~12 tRNA^His genes per haploid Arabidopsis genome by Southern analysis (data not shown), the existence of two new Arabidopsis tRNA^His genes containing C₅₄ found in two independent BAC clones (Genbank accession nos AF001308 and Z97336) and the PCR and restriction enzyme analysis, it seems likely that all Arabidopsis nuclear tRNA^His genes contain C₅₄. This exceptional nucleotide is conserved in the other three plants examined, including lupin. Therefore, it is unlikely that the original Arabidopsis tRNA^His gene is a pseudogene.

Figure 3. Alignment of PCR-amplified DNA sequences from four different dicotyledonous species with plant tRNA^His-specific primers. The top sequence is the coding region (nt 20-54) of the Arabidopsis tRNA^His gene. Numbering is as in Sprinzl et al. (6). C₅₄ is outlined. Underlined nucleotides indicate the anticodon. In the other sequences only the regions amplified between the two PCR primers are shown. Nucleotides identical to those in the top sequence are shown by a dash. Numbers in brackets indicate the number of independent clones analysed. At (a-d), Arabidopsis;Ll, lupin; Ps (a and b), pea; Nt, tobacco.

Plant cytosolic tRNAs^His contain C₅₄

To evaluate the possibility that C₅₄ is edited to U₅₄ in tRNA^His transcripts in plants we performed northern analysis of tRNA^His from Arabidopsis and lupin using several specific oligonucleotide probes (shown in Fig. 2) to discriminate between C₅₄ and U₅₄ or their derivatives. We also used Atsp and Llsp, which are complementary to nt 19-36 in the reported Arabidopsis and lupin sequences respectively, because most of the differences between these sequences are concentrated in nt 20-30 of the tRNA. Hybridization was carried out at a temperature 5°C lower for C54 and U54 and 10°C lower for Atsp and Llsp than their T_m in 1 M NaCl. Washing for both kinds of probes was done at 5°C below their T_m in 1 M NaCl. As shown in Figure 5A, probes C54 and U54 can efficiently discriminate one nucleotide difference at position 54 between an Arabidopsis tRNA^His gene with C₅₄ (tHis/wt) and a mutated tRNA^His gene with T₅₄ (tHisU54). A PCR-generated tRNA^His probe including the entire coding region of tRNA^His allowed us to detect a strong signal with the tRNAs from both plants. A detectable signal was observed with the C54 probe, while no signal was detected with the U54 probe. Furthermore, Atsp gave a stronger signal than Llsp (Fig. 5B). These results are coherent with the DNA sequence results. For further verification single-stranded cDNAs were synthesized from the tRNAs using the His3[prime] oligonucleotide in the presence or absence of AMV reverse transcriptase (RT) and subsequently double-stranded cDNAs were PCR amplified with primers His5[prime] and His3[prime]. Gel analysis of the products revealed no amplification of DNA in reactions without RT, thus eliminating the possibility of amplification from genomic DNA. The DNA samples were cloned and then 48 samples (24 each for Arabidopsis and lupin) were sequenced. All the sequences could be classed amongst the sequences shown in Figure 3, except for an A->G substitution at position 20A of two lupin clones (data not shown). Besides, no NspV sites (T₅₄TCGAA) were found in these PCR-amplified cDNA sequences (based on the results of NspV digestion analysis; data not shown). Thus, there is no evidence for post-transcriptional editing of plant tRNAs^His at position 54. In addition, we found no strong evidence for any sequences corresponding to the previously published lupin sequence.

Figure 4. Restriction enzyme analysis of the PCR products of plant tRNA^His genes. PCR products (10 ng) were digested with NspV and separated by electrophoresis on a 3% agarose gel. The gel was blotted and the blot first probed with a tRNA^His gene-specific probe (PCRHis) obtained by PCR amplification with His5[prime] and His3[prime] primers in the presence of DIG-11-dUTP. After removal of the probe it was reprobed with 3[prime]-end-labelled His3[prime] with DIG-11-ddUTP by terminal deoxynucleotidyl transferase. Samples in lanes 7-12 were cleaved with NspV. tDNA indicates the complete 72 nt PCR product. 5[prime] and 3[prime] indicate the 5[prime] and 3[prime] fragments of NspV-cleaved PCR products respectively. The template DNAs used for PCR were tHis/wt (lanes 1 and 7), tHisU54 (as a positive control, lanes 2 and 8) and DNA from Arabidopsis (lanes 3 and 9), lupin (lanes 4 and 10), pea (lanes 5 and 11) and tobacco (lanes 6 and 12).

Figure 5. Northern analysis of tRNAs from Arabidopsis (At) and lupin (Ll) plants. The amount of tRNAs loaded (µg) is indicated above each lane. The PCR His probe is as described in Figure 4. Oligonucleotide probes C54 and U54 (A) or Atsp and Llsp (B) were 3[prime]-end-labelled with DIG-11-ddUTP. Their sequences are presented in Figure 2. Two plasmid DNAs (1 or 10 ng) containing the original Arabidopsis gene (tHis/wt) or the mutated gene with T₅₄ (tHisU54) were used as controls.

In vivo aminoacylation of Arabidopsis cytosolic tRNA^His

In order to explore whether this novel tRNA^His with C₅₄ is functional we determined the proportion of tRNA^His which is acylated in vivo. Total Arabidopsis tRNAs were isolated from young seedlings under acidic conditions. They were fractionated on a polyacrylamide/urea gel, also under acidic conditions, and then transferred to a membrane and probed with a labelled oligonucleotide (C54 or U54). The principle is that aminoacylated tRNAs, especially those carrying relatively basic amino acids such as lysine, arginine or histidine, migrate slower than uncharged ones in the gel due to the presence of positive charge on their amino acid residues (21). As shown in Figure 6A, only the C54 probe resulted in a detectable signal. In unhydrolysed tRNA samples two bands were observed, while only one band was detected in pre-deacylated samples, corresponding to the faster migrating band in the untreated sample. These results demonstrate that the faster and slower migrating bands are uncharged and charged tRNA^His respectively, strongly suggesting that tRNA^His with C₅₄ is aminoacylated. Nevertheless, charged tRNA^His was estimated at only ~60% of the total tRNA^His by densitometric measurements. In parallel we calculated the proportion of charged tRNA^Gly and tRNA^Asp by the same procedure. For these tRNAs nearly 100% of the molecules were charged (Fig. 6B).

In vitro transcription of Arabidopsis nuclear tRNA^His genes

Since C₅₄ is located in the B box internal control region of eukaryotic tRNA genes, one might suppose that this may impair transcription. To explore this in vitro transcription of the wild-type tRNA^His gene was compared with that of a mutated tRNA^His gene (tHisU54) with T₅₄ in place of C₅₄. The in vitro transcription system used in this study was first established by Fan and Sugiura (22) and optimized for plant nuclear tRNA genes by Yukawa et al. (19). Template DNAs were purified and their concentrations were precisely determined. DNA and tobacco nuclear extract were prepared and incubated in the presence of [[alpha]-³²P]UTP at 28°C for 2 h. Transcripts were fractionated on a denaturing polyacrylamide gel. As shown in Figure 7, both template DNAs resulted in transcripts of the expected size (75 nt), indicating that both are transcribed and processed faithfully. tHisU54 reproducibly displayed over 5-fold higher transcriptional activity than tHis/wt. These results demonstrate that the presence of C₅₄ in the wild-type Arabidopsis tRNA^His gene does not influence tRNA processing but does decrease transcriptional efficiency.

Figure 6. Detection of in vivo aminoacylation of Arabidopsis tRNAs. Total tRNAs were isolated from young Arabidopsis seedlings under acidic conditions (pH 5.0). They were electrophoresed on a polyacrylamide/urea gel containing sodium acetate, pH 5.0, and then transferred to membranes. Hybridization was carried out using two different DIG-labelled probes, C54 and U54, in (A) and two different nuclear gene sequences for Arabidopsis nuclear tRNA^Gly (5[prime]-GCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACA-GACCCGGGTTCGATTCCCGGCTGGTGCA-3[prime]; Akama, unpublished results) and tRNA^Asp (5[prime]-GTCGTTGTAGTATAGTGGTAAGTATTCCCGCCTGTCACGCGGGTGACCCGGGTTCGATCCCCGGCAACGGCG-3[prime]; 35), produced by PCR with their specific primer sets in the presence of DIG-dUTP, in (B). Ch and Un indicate charged and uncharged tRNAs respectively. The intensity of bands was measured with a densitometer. Values of charged and uncharged tRNAs are presented as a percentage of both tRNAs. (+) and (-) indicate that the tRNAs were or were not deacylated before loading respectively. n.d., not determined.

In vivo transcription of an Arabidopsis nuclear tRNA^His gene with an anticodon complementary to an amber codon (UAG)

In an attempt to verify expression and function of tRNA^His with C₅₄ in plant cells we examined expression of tHis/amber in transformed calli. The tHis/amber clone, constructed from an Arabidopsis tRNA^His gene by site-directed mutagenesis, carries a modified anticodon (CTA) complementary to the amber stop codon. Due to the 2 nt changes in the anticodon the transcript expressed from tHis/amber can be detected by a specific oligonucleotide probe. The mutated gene was subcloned into the T-DNA region of a Ti plasmid vector carrying a gene conferring resistance to kanamycin and introduced into Arabidopsis cells via Agrobacterium. Calli resistant to kanamycin were obtained 2 weeks after selection and tRNAs were isolated. Figure 8 indicates the results of northern hybridization analysis using the probes His3[prime] and C34A36. The His3[prime] probe gives a signal of the same intensity on RNAs from both cell lines. In contrast, the C34A36 probe hybridizes much more strongly to tHis/amber RNA than to tHis/wt RNA. These results confirm that a nuclear tRNA^His gene with C₅₄ is transcriptionally active in plant cells. Using a [beta]-glucuronidase marker gene carrying a premature stop codon we hoped to be able to show that this C₅₄-containing tRNA^His suppressor tRNA was functional in protein synthesis, but unfortunately no amber suppressor activity could be detected with the tHis/amber construct. A similar result was obtained with amber suppressor derivatives of plant tRNA^Phe (23), where it was shown that the alterations to the anticodon prevented aminoacylation. It seems likely that a similar problem precludes the use of tRNA^His-derived amber suppressors. In addition, it was shown for yeast tRNA^His that the correct anticodon is a prerequisite for histidinylation (24).

DISCUSSION

It is clear from these results that plant nuclear tRNA^His genes with a C₅₄ instead of the usual T₅₄ present in other tRNA genes are common in plant genomes and in fact we found no solid evidence that tRNA^His genes containing T₅₄ variants exist in plants. These C₅₄-containing genes are transcribed in vitro and in vivo and C₅₄-containing tRNA^His is aminoacylated in vivo as judged by acid gel electrophoresis, albeit we did not prove histidinylation. The C₅₄ is not changed to a U after transcription and although the base may be modified in some way, if it is, the modified base is read as C by reverse transcriptase. Usually the nucleotide at position 54 in cytosolic tRNAs is a methylated derivative of uridine or pseudouridine, which would have been read as U if present. Our lupin tRNA^His sequences resemble the Arabidopsis tRNA^His gene sequences and differ from the previously reported sequence of cytosolic lupin tRNA^His (8). We can only assume that this reflects errors in the original lupin sequence, given the much greater technical difficulties with direct RNA sequencing. Unfortunately, the data for regions of the lupin tRNA which apparently differ from our sequences are not presented in Barciszewska et al. (8).

Figure 7. In vitro transcription analysis of Arabidopsis wild-type and modified tRNA^His genes in tobacco nuclear extract. 0.1 pmol each template DNA and reaction mixtures containing 30 µg crude nuclear tobacco proteins were assembled and subsequently incubated at 28°C for 2 h. Transcripts were fractionated on a 12% polyacrylamide/8 M urea gel. The arrow indicates the size of the mature tRNA transcripts. Size marker, pBR322 cleaved with HaeIII; w/o DNA, no DNA added; tHis/wt, wild-type Arabidopsis tRNA^His gene; tHisU54, modified tRNA^His gene with T₅₄ in place of authentic C₅₄.

In the three-dimensional crystal structure of yeast tRNA^Phe (25) T₅₄ and A₅₈ form a reversed Hoogsteen pair that maintains the sharp bend in the T[Psi]C loop at the `corner' of the L-shaped tRNA molecule and this important function presumably explains the high conservation of the these two bases (5). Only a few exceptions to the general rule of an invariant T₅₄ have been reported so far. Eukaryotic initiator tRNAs contain an A₅₄ (6), which, like the usual T₅₄, is hydrogen bonded to A₅₈ (26). The small difference in structure may explain why initiator tRNA binds preferentially to eIF2 rather than EF-1[alpha] and neither a U₅₄ nor a U₅₄C₆₀ initiator tRNA mutant in yeast can support cell growth (27), although double mutations (U₅₄U₆₀) in human initiator tRNA have much less effect on its function (28). Several insect tRNA^Ala genes also contain A₅₄, as do a few isolated examples of other tRNAs from other organisms. The significance of this nucleotide in these tRNAs has not been examined. It should be noted that the plant tRNAs^His described here are the first natural tRNAs to be shown to contain C₅₄. This nucleotide cannot hydrogen bond to A₅₈ in the same manner as U₅₄, T₅₄ or A₅₄ and thus the structure of the T[Psi]C loop in this tRNA is difficult to predict. In this context it is worth noting that a yeast initiator tRNA mutant with C₅₄ is still functional (27) and a Drosophila tRNA^Arg mutant with C₅₄ is processed normally (29), indicating that the tertiary structure of the tRNA is unlikely to be greatly perturbed and/or it is likely to be able to mimic normal tRNA structure.

Figure 8. Detection of Arabidopsis tRNA^His (GUG) and tRNA^His (CUA) in transgenic Arabidopsis tissues. Transformed calli carrying the tHis/wt or tHis/amber gene constructs were produced by an Agrobacterium-mediated procedure (18). tRNAs (5, 10 or 15 µg) extracted from calli were loaded on a 10% polyacrylamide/8 M urea gel and then transferred to a membrane. The filter was first probed with labelled His3[prime]. After removal of the probe it was reprobed with labelled C34A36. These steps were repeated using the same probe. Hybridization was performed in 6× SSC at 42°C overnight. Washing was first in 6× SSC, 0.1% SDS at room temperature and then in the same buffer at the indicated temperature (Temp) twice. Immunochemical detection of hybridized probes was as described in Materials and Methods. As a control a Southern blot filter of 10 or 100 ng plasmids tHis/amber and tHis/wt, digested with BssHII, was probed in parallel.

Transcription of eukaryotic nuclear tRNA genes is controlled by the A and B boxes of the internal promoter, which correspond to the D stem and loop and T[Psi]C stem and loop in the tRNA respectively (reviewed by 1-4). Several sequences in these boxes are highly conserved and studies have shown that mutations in these regions generally result in a drastic reduction in transcription (30,31). However, point mutations (T->C or A) at position 54 of the B box of a Drosophila tRNA^Arg gene have much less effect on transcription than mutations of the surrounding nucleotides, both in homologous and heterologous (HeLa) nuclear extracts (29). Thus variation at position 54 may be better tolerated than at other nucleotides in the B box. Nevertheless, in our system C₅₄ does seem to negatively affect transcription rate. Acquisition of C₅₄ might have naturally become fixed in plants by chance, because there is no lethal effect on the cells. Alternatively, one might speculate that plants have gained a unique function at the sacrifice of reduced transcription and aminoacylation efficiency of cytosolic tRNA^His. With this in mind, two interesting findings related to tRNA^His are worth considering. Ciechanover et al. (32) have suggested that tRNA^His, probably the uncharged form, regulates the ubiquitin- and ATP-dependent proteolytic system in mammalian cells. More recently Zhu et al. (33) have shown in yeast that uncharged tRNA^His activates the protein kinase GCN2 (which contains a histidyl-tRNA synthetase-like domain) to phosphorylate eIF2. This in turn leads to a decrease in translation initiation and activates a set of genes involved in amino acid biosynthesis. It is highly likely that all cells contain similar sensing mechanisms to regulate amino acid biosynthesis, translation and protein turnover, based on charging levels of particular tRNAs, in response to cellular demand. The naturally low level of charging of Arabidopsis tRNA^His shown by acid gel electrophoresis (Fig. 6A) compared with other tRNAs would make it a particularly sensitive marker.

ACKNOWLEDGEMENTS

We would like to thank Dr Shigeyuki Tanifuji for his encouragement and suggestions and Jan Drouaud for his efficient sequencing. This work was partly supported by a Grant-in-Aid from the Ministry of Education, Japan.

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*To whom correspondence should be addressed. Tel: +81 852 32 6431; Fax: +81 852 32 6429; Email: akama@life.shimane-u.ac.jp

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