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Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro
Introduction
Materials And Methods
Reagents
DNA plasmids and synthetic tRNA substrates
Preparation of P.furiosus S100 extract
In vitro enzymatic assay
Analysis of modified nucleotides
Results
Analysis of the modified nucleotides formed in tRNA incubated with P.furiosus cell-free extract
Modified nucleotides located in the T[Psi] loop are insensitive to the three dimensional (3D) architecture of the tRNA substrate
m1A57 is an intermediate in m1I57 formation
Discussion
Acknowledgements
References
Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro
ABSTRACT
INTRODUCTION
Naturally occurring tRNAs always contain a variety of chemically altered nucleotides (at least 80 different structures reported to date; 1), which are formed by enzymatic modification of the primary RNA transcript during the sequential process of tRNA maturation. Most of these nucleotides bear simple modifications such as ribose or base methylations, base isomerization, base reduction, base thiolation or base deamination. Other naturally occurring modified nucleotides represent more complex modifications such as group additions or multiple modifications on a base and/or on the ribose. These hypermodified residues are most frequently found at position 34 (the first anticodon base) and at position 37 (3[prime]-adjacent to the third anticodon base) of the tRNA anticodon loop (reviewed in 2).
So far, tRNA modification enzymes have been identified and characterized biochemically mostly in bacteria (Escherichia coli, Bacillus subtilis, Bacillus stearothermophilus,...), in yeast(Saccharomyces cerevisiae, Shizosaccharomyces pombe,...) and in higher eukaryotes (rat, human placenta,...). In contrast, only a few studies have dealt with tRNA modification enzymes in extracts of archaea (for reviews see 3,4).
In the pioneering work of Best (5), the cell extract of Methano-coccus vannielii supplemented with labelled [3H-CH3]AdoMet was used to reveal the activities of several tRNA:methyltrans-ferases catalyzing the formation of methylated nucleotides (m1A, m1G, m2G, m22G and Cm) in undermethylated bulk E.coli tRNA used as substrate. No m7G or m5U were detected, which is consistent with the absence of these modified residues in naturally occurring M.vannielii tRNA, implying that the corresponding methyltransferases were absent. In our previous work, the T7 transcript of Haloferax volcanii tRNAIle was used to reveal the enzymatic activities of the modification enzymes specific for the formation of m22G, m5C, [Psi], m1[Psi], m1A, m1I and Cm present in the cell extracts of H.volcanii (6). Recently, the activity of a novel tRNA-guanine transglycosylase catalyzing the formation of the modified base archaeosine in position 15 of archaeal tRNA has been demonstrated in H.volcanii extract using homologous tRNAs (7; see also 8). Also, the gene for the tRNA(guanine-26, N2-N2) methyltransferase (Trm1) from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and successfully expressed in an active form in E.coli; this recombinant enzyme was shown to specifically catalyse the in vitro formation of m22G at position 26 in the T7 transcript of yeast tRNAPhe (9).
Pyrococcus furiosus is a sulphur-metabolizing hyperthermophile of the order Thermococcales which grows optimally at 100°C under strictly anaerobic conditions (10). So far, none of the tRNA species from this archaeal organism, or from other hyperthermophilic archaea, have been directly sequenced. However, the occurrence of modified nucleotides in bulk tRNA from P.furiosus was demonstrated by high performance liquid chromatography combined with mass spectrometry (LC/ESI-MS; 11). It was shown that P.furiosus tRNA contains several typically eukaryotic modified nucleotides, like Am, m1I, m5C, m2G and m22G. In addition, tRNA molecules from this hyperthermophilic archaeon also contain a set of unique hypermodified nucleotides, such as 2[prime]-O-methyl derivatives of m22G and ac4C as well as the 2-thiolated derivative of m5U. However, their locations and distribution within individual species of P.furiosus tRNA are not known.
The identification and comparison of enzymes that catalyse the modification of the nucleotides in tRNA of different organisms may shed light on the evolutionary origin of the modification machinery. Also, the study of cross-reactions between enzymes and tRNAs from different phylogenetic groups should inform on the conservation of the identity elements that are required for modification of a given nucleotide in tRNA. This idea is reminiscent of a similar approach that was successfully used to study aminoacyl-tRNA synthetases and tRNA recognition by these enzymes (reviewed in 12,13).
In this work we used T7 transcripts of two yeast tRNA genes (namely tRNAAsp and tRNAPhe) and of one tRNA gene (tRNAIle) of H.volcanii to characterize several tRNA modification enzymes from P.furiosus, as well as to locate their potential sites of modification in heterologous tRNAs.
MATERIALS AND METHODS
Reagents
[[alpha]-32P]-radiolabelled nucleotide triphosphates (400 Ci/mmol) were from Amersham (UK). Penicillium citrinum nuclease P1 and Aspergillus oryzae RNase T2 were from Sigma (St Louis, MO), S-adenosyl-l-methionine (AdoMet) from Boehringer Mannheim (Mannheim, Germany). Chemically synthesized deoxyoligonucleotides were purchased from MWG-Biotech (Ebersberg, Germany) and used without further purification. Thin layer cellulose plates (TLC) (type F1440) were from Schleicher and Schuell (Dassel, Germany). All other chemicals were from Merck Biochemicals (Darmstadt, Germany).
DNA plasmids and synthetic tRNA substrates
All plasmids bearing various tRNA genes used in this study are described elsewhere: yeast wild-type tRNAPhe(GAA) and its variant (G10->C, C25->G) (14,15); wild-type yeast tRNAAsp(GUC) and its variants (16,17); wild-type H.volcanii tRNAIle(GAU) and its mutant A57->G (6). The corresponding tRNA transcripts, lacking all the nucleotide modifications and separately 32P-radiolabelled with each one of the four nucleotides, were prepared by in vitro transcription using T7 RNA polymerase, following the procedure described previously (18). The minihelices and most of the T[Psi] stem-loop variants of tRNAAsp were prepared by in vitro transcription as described (19).
Preparation of P.furiosus S100 extract
Cell-free extract of P.furiosus (strain DSM 3638) grown at 98°C under strictly anaerobic conditions as described (10; see also 20) was prepared in 25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10% glycerol, 2 mM DTT, 1 mM PMSF, 1 mM DFP, 1 mM benzamidine by sonication followed by ultracentrifugation at 100 000 g for 1 h. The S100 extract was stored frozen at -20°C in the presence of 20% glycerol. The protein concentration was determined according to Bradford (21).
In vitro enzymatic assay
The standard assay mixture (total volume 50 µl) contained 25 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, 2 mM DTT, 75 µM AdoMet, 20 U RNasin and 40 fmol 32P-radiolabelled tRNA transcript (~5 × 104 c.p.m.). The reaction was initiated by the addition of the protein extract (1 mg/ml final concentration). Incubations were performed at 50°C for 1 or 2 h as specified in figure legends. To prevent evaporation, the reaction mixture was overlayed with 10 µl of paraffin oil (PCR oil). After incubation, the proteins were removed by phenol/chloroform extraction and the radiolabelled transcript was recovered by ethanol precipitation.
The stability of T7 tRNA transcripts towards endonucleolytic degradation in the P.furiosus S100 extract was also verified. It was noticed that, under the conditions of the test, the tRNA substrates are sufficiently stable since <20% of the transcripts were degraded during the incubation for 60 min (revealed by gel electrophoresis and quantified by trichloroacetic acid precipitation; data not shown).
Analysis of modified nucleotides
Modified tRNA transcripts were digested overnight by nuclease P1 (2 µg) in 50 mM ammonium acetate buffer at pH 5.3 or by RNase T2 (0.1 U) in the same buffer at pH 4.6. Identification of the 32P-labelled nucleotides in tRNA hydrolysates was performed by two-dimensional (2D) chromatography on TLC plates. To unam-biguously identify each modified nucleotide on the TLC plates by comparison with reference maps (22), two chromatographic systems were used: in both cases, the first dimension was developed with isobutyric acid:25% ammonia:water (66:1:33 by volume), while the second dimension was developed in 0.1 M sodium phosphate, pH 6.8:solid ammonium sulfate:n-propanol for the system N/R (100:60:2 v/w/v) and with 2-propanol:37% HCl:water (68:17.6:14.4 v/v) for the system N/N. Radioactive spots were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) with software ImageQuant. Calculation of the molar amount of modified nucleotide per mol tRNA substrate was done by taking into account the nucleotide composition of the tRNA transcript. For a given batch of enzyme extract, the experimental error was determined by triplicate analysis of identically treated tRNA transcripts and found to be ~±0.1 mol/mol tRNA.
RESULTS
Analysis of the modified nucleotides formed in tRNA incubated with P.furiosus cell-free extract
Figure
Figure 1. (A) Compilation of the modified nucleotides found in the 59 sequenced archaeal tRNAs indicating type, location and frequency of each modification. Abbreviations of modified nucleotides are according to Sprinzl et al. (23). Numbers in the cloverleaf correspond to location in tRNA. The first number next to each symbol in square boxes indicates the frequency of occurrence of a particular modified nucleotide; the second number corresponds to the frequency of the parent nucleotide U, C, G or A. For example, at position 8 of tRNA, s4U is found only once over a total of 59 archaeal tRNAs bearing a uridine at position 8. Unique modified nucleotides found only in archaeal tRNAs (both by chemical structure and location in the molecule) are shaded. (B-D) Cloverleaf structures of naturally occurring tRNA molecules, the T7 transcripts of which were used as substrates in in vitro tests. Modified nucleotides found in the naturally occurring tRNAs (i.e. homologous modifications) are boxed. Those modified nucleotides that are also found in archaeal tRNA are shaded. Each of the T7 transcripts corresponding to yeast tRNAAsp, yeast tRNAPhe and H.volcanii tRNAIle was separately radiolabelled with all four [[alpha]-32P]nucleoside triphosphates (ATP, CTP, GTP or UTP). Each labelled transcript was then incubated with dialysed cell-free P.furiosus extract (S100) supplemented with AdoMet. After incubation, the radiolabelled modified tRNAs were analysed for the presence of modified nucleotides as described in Materials and Methods. The results derived from the analysis of three tRNA substrates are summarized in Table 1. Figure 
The results indicate that several modified nucleotides were synthesized in tRNA transcripts upon the incubation with P.furiosus S100 extract. Six chemically different modified residues were formed in yeast tRNAAsp, seven in yeast tRNAPhe and nine in H.volcanii tRNAIle. They correspond to the following different types of modified nucleotides: 2[prime]-O-methyladenosine (Am), N2-methylguanosine (m2G), N2,N2-dimethylguanosine (m22G), N1-methylguanosine (m1G), 5-methylcytosine (m5C), pseudouridine ([Psi]), ribothymidine (T), 2[prime]-O-methylcytosine (Cm), N1-methylinosine (m1I) and N1-methyladenosine (m1A). In addition to the modifications expected from the sequence of the modified tRNAs derived from homologous systems, all tRNA transcripts bear some additional modified residues specific for the P.furiosus modification machinery (indicated in bold in Table 1). The yeast tRNAAsp transcript has four such additional modifications (m1A, m1I, Cm and m2G), tRNAPhe has one (Cm) and tRNAIle has five (Cm, m1A, m2G, m22G and Am). It is noteworthy that the Cm residue in tRNAPhe is formed not at the expected position 32 but at position 56, which is a characteristic feature of archaeal tRNAs.
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Table 1.
The locations of modified nucleotides in the tRNA transcripts were deduced from the known modification patterns of yeast and archaeal tRNAs (Fig.
In the case of the m2G/m22G modifications one cannot decide if they are present at positions 10 and/or 26 in yeast tRNAPhe and in H.volcanii tRNAIle, since both residues have the same 3[prime]-nearest neighbour nucleotide (CMP). In addition, m2G is a reaction intermediate of m22G formation. Optimization of reaction conditions in the case of tRNAPhe allowed detection of 1.1 mol/mol of m2G and thus indicates the possibility of modification at two independent sites. On the other hand, only formation of m22G was detected in mutant tRNAPhe (G10->C, C25->G), where the other potential modification site (G10) is absent (9; data not shown). Taken together these results allow us to conclude that m2G is formed at position 10 and m22G at position 26 in tRNAPhe and tRNAIle. This conclusion also agrees with the detection of m2G10 in the tRNAAsp transcript. A similar situation is found for m5C at positions 40 and 49 in H.volcanii tRNAIle bearing GMP as nearest neighbour nucleotides. Since m5C is quantitatively formed at position 49 in yeast tRNAAsp, one may expect that the same residue would also be modified in other tRNA transcripts. However, in yeast tRNAPhe, no m5C was detected (neither at position 40 nor 49).
Figure 2. Selected examples of 2D TLC separation of modified nucleotides formed in different tRNA transcripts during incubation at 50°C with cell-free P.furiosus extract. Analysis of modified nucleotides by TLC was performed in chromatographic system N/R (Materials and Methods), except the one presented in (E) where system N/N was used. The type of tRNA transcript, the nature of 32P label and the nuclease used for digestion are indicated in each panel. The radioactive spots, as revealed by autoradiography, correspond to parental nucleotides A, C, G and U, inorganic phosphate (pi) and to detected modified nucleotides. Empty circles show the positions of non-labelled nucleotide monophosphate markers, detected by UV shading. Analysis of the modification pattern of H.volcanii tRNAIle by T2 hydrolysis is more difficult, especially for modified nucleotides at positions 56-58. Depending on the modification efficiency, one can detect several modified dinucleotides after T2 digestion (Fig. The position of Am formation in H.volcanii tRNAIle was deduced from the results of T2 digestion, where this modified residue was found 5[prime]-adjacent to A. Four potential AA sites are present in tRNAIle (positions 6-7, 37-38, 57-58 and 58-59). The results of nearest neighbour analysis indicate that the modification occurs in the AAU sequence in tRNAIle (Table 1, UTP/T2 labelling/digestion). This trinucleotide is present at positions 6-8 and positions 58-60. The latter location can be excluded since a considerable amount of another modified base (m1A58) is synthesized at this site and almost 0.9 mol/mol of AmA (Table 1) was detected in the tRNAIle transcript. Similar results were also obtained for the A57->G tRNAIle variant where m1A58 formation is quantitative (see above). On the other hand, the formation of the same AmA dinucleotide was detected in yeast tRNAVal which has the only similar AA sequence at positions 6-7 (data not shown). These results allowed us to conclude that Am is formed at position 6 in H.volcanii tRNAIle. Figure Figure 3. Modification patterns of yeast tRNAAsp, tRNAPhe and H.volcanii tRNAIle incubated in P.furiosus S100 extract. Potential modification sites are boxed, modified nucleotides formed in tRNA transcripts are shaded. Modified nucleotide m2G is most probably formed at position 10 in H.volcanii tRNAIle (boxed, but not shaded), but due to the same neighbouring nucleotide it cannot be distinguished from the reaction intermediate of m22G formation at position 26. The enzymatic formation of at least two modified nucleotides located in the T[Psi] loop of E.coli or yeast tRNA (ribothymidine 54 and pseudouridine 55) was shown to be independent of the global 3D structure of tRNA and efficiently occurred in the minisubstrates comprised of only the T[Psi] stem-loop structure (19,25,26). In order to verify whether the same conclusion applies to the enzymes from P.furiosus, as well as to test for the enzymatic formation of other modified nucleotides, several tRNAAsp mutants with stepwise reduction of the tRNA size (deletion of D stem-loop, minihelix containing the acceptor stem and T[Psi] stem-loop as well as shorter minihelices) were used as substrates (Figure Table 2. Incubation of the [32P]ATP-labelled H.volcanii tRNAIle with P.furiosus extract gave two characteristic dinucleotide diphosphates after hydrolysis with RNase T2, one corresponding to Cmm1Ap and the other to the dinucleotide diphosphate Cmm1Ip (Fig. 
Modified nucleotides located in the T[Psi] loop are insensitive to the three dimensional (3D) architecture of the tRNA substrate
m1A57 is an intermediate in m1I57 formation
DISCUSSION
Run-off transcripts of synthetic genes with the primary structure of a natural tRNA but lacking all modified nucleotides have been proven to be valuable substrates for testing the activity of several tRNA modification enzymes in vitro (reviewed in 27). For testing the activity of enzymes present in the cell-free extract of P.furiosus, we used the transcripts corresponding to yeast tRNAAsp, yeast tRNAPhe and H.volcanii tRNAIle as a model system. The modification pattern of these tRNAs was extensively characterized under various assay conditions (28-30). Also, the physicochemical properties of the yeast tRNAPhe and yeast tRNAAsp transcripts are known (14,16) and several mutants with disrupted 3D tRNA structure are available (15,17,29). The use of these heterologous tRNAs as substrates for P.furiosus enzymes was based on the expectation that they may also serve as substrates in heterologous reaction. Indeed, the modification pattern of archaeal tRNA (Fig.
Figure 4. Structures of tRNAAsp minisubstrates used for detection of modified nucleotides synthesized in the T[Psi] loop of tRNA. Locations of the modified nucleotides formed in each minisubstrate are indicated by arrows. All quantitative data are summarized in Table 2. Domain I of tRNA (acceptor stem extended by T[Psi] stem-loop) is boxed. In this work, the heterologous transcripts of yeast and H.volcanii tRNAs were used to test the activity of P.furiosus modification enzymes in vitro. The incubation temperature was fixed at 50°C, instead of 70-100°C corresponding to optimal cell growth. This temperature was chosen taking into account the melting of unmodified T7 transcripts of tRNAPhe and tRNAAsp, which begins at 55-60°C (14,16). However, this elevated incubation temperature may nevertheless affect folding of unmodified heterologous tRNA transcripts in the extract and somehow hamper the activity of tRNA 3D structure-dependent modification enzymes. The other major limitation of the in vitro approach lies in the lack of knowledge on low molecular mass reaction cofactors that are required for enzymatic formation of certain ‘complex’ modified nucleotides (for example t6A). The use of heterologous tRNA transcripts has allowed us to identify the activities of 11 modification enzymes present in the P.furiosus cell-free extract. Most of the detected modifications correspond to AdoMet-dependent methylations of base (m5C, m2G, m22G, m1G, m1A and m1I, T) or ribose (Am and Cm) and formation of pseudouridine at two distinct positions ([Psi]39 and [Psi]55) in tRNA. The locations of most of the synthesized modified residues are similar to those observed for yeast tRNAs (m22G26, m1G37, [Psi]39, m5C49, T54, [Psi]55 and m1A58). However, some archaea-specific modifications present in the tRNA T[Psi] loop (Cm56 and m1I57) and in the aminoacyl acceptor stem (Am6) were also detected. Ribose-methylated adenosine has not been found at position 6 (Am6) in any tRNA sequenced so far, but the same modified nucleotide was detected at position 4 (Am4) in a few eukaryotic tRNA (23). Several modified nucleotides ([Psi], m5C and Cm) can be formed at multiple sites in archaeal tRNA. Two potential sites of m5C methylation (positions 40 and 49) are present in H.volcanii tRNAIle. Our data suggest that only one m5C residue is formed in this transcript upon incubation in the P.furiosus extract. Most probably the modification takes place at position 49 while position 40 remains unmodified. In yeast tRNAPhe the formation of m5C40 is a strictly intron-dependent process and the pre-tRNA structure is required for modification (30). In contrast, the tRNAIle gene of H.volcanii has no intervening sequence (R.Gupta, unpublished results). On the other hand, it was recently demonstrated that the formation of Cm32 and Cm34 in H.volcanii tRNATrp depends on the presence of an intron (C.Daniels, reported at the 98th ASM annual meeting in Atlanta, May 1998). Hence, it is possible that the absence of Cm32 formation in tRNAPhe is related to the use of an intronless tRNA transcript. The biosynthesis of [Psi]13, [Psi]38/39 and [Psi]55 is catalyzed by distinct enzymes in E.coli and yeast. Our results demonstrate that [Psi]39 and [Psi]55 are efficiently synthesized in P.furiosus extract. In contrast, the formation of [Psi]13 in the yeast tRNAAsp transcript was not detected, while the formation of this residue occurs upon incubation in yeast extract (31). Modification of [Psi]13 is a characteristic feature of the archaeal tRNAs (out of 30 uridines at position 13 only one remains unmodified; Fig. In our in vitro study we detected the formation of ribothymidine (m5U, T) at position 54 in all three tRNA transcripts. It should be noted that this modified nucleoside is highly conserved in prokaryotic and eukaryotic tRNAs, yet is only rarely present in archaeal tRNA. In the majority of the sequenced tRNAs it is replaced by m1[Psi] or hypermodified derivatives of T (such as s2T or s2Tm). However, the HPLC analysis of P.furiosus tRNA extracted from cells grown at different temperatures reveals the presence of T in cells grown at 50-60°C, while it is completely absent at 95-100°C (only s2T or s2Tm are present) (11). The efficient formation of T54 in vitro most probably reflects the in vivo biosynthetic pathway, but due to the absence of 2-thiolation, the reaction yields only the intermediate which normally does not accumulate. The absence of detectable amounts of inosine formed at position 57 in tRNA (as an intermediate of m1I57 biosynthesis) is noteworthy. While inosine is the intermediate of m1I formation at position 37 in yeast tRNAAla (34), the pathway of m1I synthesis in Archaea is different. It has been demonstrated that in H.volcanii tRNA, m1I57 is formed by deamination of m1A57 and not by the methylation of inosine 57 (6). Here we extend this conclusion to P.furiosus and S.shibatae. Taken together these results allow us to conclude that the pathway of m1I synthesis in Archaea is different from the one found in Eucaryota. Numerous studies have clearly established the existence of three major domains of life (Archaea, Bacteria and Eucaryota) (35). However, detailed studies of the archaeal metabolism and enzymes strongly indicate the presence of both eubacterial and eukaryotic features (36-40). The basal metabolism of archaeal cells is more closely related to that of eubacteria, while the metabolism related to gene expression (DNA transcription, mRNA synthesis, mRNA translation and splicing) is definitely more related to that of eukaryotes. The reactions implicated in tRNA modification occupy an intermediate position. These processes are tightly connected to basal cellular metabolism as they use numerous low molecular weight cofactors, synthesized also for other purposes (AdoMet, sulphur-containing precursors, isopentenyl-pyrophosphate,...). On the other hand, since tRNA molecules play a key role in the process of mRNA decoding, one can also expect that the tRNA modification machinery is related to that of Eucaryota, which is indeed the case for formation of m2G10, m22G26 and m5C49. In addition, the archaeal tRNA modification machinery has several unique features (the presence of Am6, Cm56 and m1I57) which clearly distinguish it from the other two living domains.
ACKNOWLEDGEMENTS
We thank Marc Demarez from the CERIA in Brussels, Belgium, for providing us with P.furiosus. We also thank Prof. P. Forterre (Université d’Orsay, France) and Dr J.-P. Waller (CNRS, Gif-sur-Yvette, France) for discussions, encouragement and critical reading of the manuscript. This work was performed in the frame of the programme Bactocean sponsored by CNRS-IFREMER. It was supported by grants from the CNRS (ACC-5 and PCV) and Actions de Recherche sur le Cancer (ARC). F.C. is a pre-doctoral fellow supported by a fellowship from the Ministère de la Recherche et de l’Enseignement Supérieurs and Actions de Recherche sur le Cancer (ARC). Y.M. is supported by an Associated Researcher position at the CNRS (Poste Rouge).
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