Portal de Periódicos da CAPES

Trm7p catalyses the formation of two 2'-O-methylriboses in yeast tRNA anticodon loop

2002; Springer Nature; Volume: 21; Issue: 7 Linguagem: Inglês

10.1093/emboj/21.7.1811

1460-2075

Lionel Pintard, François Lecointe, Janusz M. Bujnicki, Claire Bonnerot, Henri Grosjean, Bruno Lapeyre,

Biochemical and Molecular Research

Article1 April 2002free access Trm7p catalyses the formation of two 2′-O-methylriboses in yeast tRNA anticodon loop Lionel Pintard Lionel Pintard Centre de Recherche de Biochimie Macromoléculaire du CNRS, 1919 Route de Mende, F-34293 Montpellier, cedex 5, France Present address: Swiss Institute For Experimental Cancer Research, Epalinges, Switzerland Search for more papers by this author François Lecointe François Lecointe Laboratoire d'Enzymologie et Biochimie Structurales CNRS, Gif sur Yvette, France Search for more papers by this author Janusz M. Bujnicki Janusz M. Bujnicki International Institute of Molecular and Cell Biology, Warsaw, Poland Search for more papers by this author Claire Bonnerot Claire Bonnerot Centre de Recherche de Biochimie Macromoléculaire du CNRS, 1919 Route de Mende, F-34293 Montpellier, cedex 5, France Search for more papers by this author Henri Grosjean Henri Grosjean Laboratoire d'Enzymologie et Biochimie Structurales CNRS, Gif sur Yvette, France Search for more papers by this author Bruno Lapeyre Corresponding Author Bruno Lapeyre Centre de Recherche de Biochimie Macromoléculaire du CNRS, 1919 Route de Mende, F-34293 Montpellier, cedex 5, France Search for more papers by this author Lionel Pintard Lionel Pintard Centre de Recherche de Biochimie Macromoléculaire du CNRS, 1919 Route de Mende, F-34293 Montpellier, cedex 5, France Present address: Swiss Institute For Experimental Cancer Research, Epalinges, Switzerland Search for more papers by this author François Lecointe François Lecointe Laboratoire d'Enzymologie et Biochimie Structurales CNRS, Gif sur Yvette, France Search for more papers by this author Janusz M. Bujnicki Janusz M. Bujnicki International Institute of Molecular and Cell Biology, Warsaw, Poland Search for more papers by this author Claire Bonnerot Claire Bonnerot Centre de Recherche de Biochimie Macromoléculaire du CNRS, 1919 Route de Mende, F-34293 Montpellier, cedex 5, France Search for more papers by this author Henri Grosjean Henri Grosjean Laboratoire d'Enzymologie et Biochimie Structurales CNRS, Gif sur Yvette, France Search for more papers by this author Bruno Lapeyre Corresponding Author Bruno Lapeyre Centre de Recherche de Biochimie Macromoléculaire du CNRS, 1919 Route de Mende, F-34293 Montpellier, cedex 5, France Search for more papers by this author Author Information Lionel Pintard1,2, François Lecointe3, Janusz M. Bujnicki4, Claire Bonnerot1, Henri Grosjean3 and Bruno Lapeyre 1 1Centre de Recherche de Biochimie Macromoléculaire du CNRS, 1919 Route de Mende, F-34293 Montpellier, cedex 5, France 2Present address: Swiss Institute For Experimental Cancer Research, Epalinges, Switzerland 3Laboratoire d'Enzymologie et Biochimie Structurales CNRS, Gif sur Yvette, France 4International Institute of Molecular and Cell Biology, Warsaw, Poland ‡L.Pintard and F.Lecointe contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1811-1820https://doi.org/10.1093/emboj/21.7.1811 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The genome of Saccharomyces cerevisiae encodes three close homologues of the Escherichia coli 2′-O-rRNA methyltransferase FtsJ/RrmJ, designated Trm7p, Spb1p and Mrm2p. We present evidence that Trm7p methylates the 2′-O-ribose of nucleotides at positions 32 and 34 of the tRNA anticodon loop, both in vivo and in vitro. In a trm7Δ strain, which is viable but grows slowly, translation is impaired, thus indicating that these tRNA modifications could be important for translation efficiency. We discuss the emergence of a family of three 2′-O-RNA methyltransferases in Eukaryota and one in Prokaryota from a common ancestor. We propose that each eukaryotic enzyme is located in a different cell compartment, in which it would methylate a different RNA that can adopt a very similar secondary structure. Introduction One of the long-term goals of modern biology is to predict the function of a protein by analysing its amino acid sequence and, whenever possible, by inspection of its three-dimensional architecture (Burley, 2000). This can be achieved by a combination of structural genomics and bioinformatics approaches, followed by testing the predicted function in vivo and in vitro. With the completion of the sequencing projects for a growing number of organisms, it is now possible to carry out genome-wide searches for genes encoding putative proteins that share sequence motifs and thus potentially belong to the same family (Goffeau et al., 1996; Koonin et al., 1998). For instance, methyltransferases (MTases) that use S-adenosyl-L-methionine (AdoMet) as a cofactor exhibit conserved motifs that map onto the catalytic face of the common fold (Fauman et al., 1999). Recently, a structural analysis has revealed that the heat shock protein FtsJ/RrmJ of Escherichia coli exhibits the typical MTase fold (Bügl et al., 2000). It has been demonstrated that FtsJ/RrmJ actually catalyses the formation of Um2552, which is located within a loop of five nucleotides present on domain V of the peptidyl transferase centre of the 23S rRNA (Caldas et al., 2000a). In vitro, FtsJ/RrmJ is able to methylate the 23S rRNA when it is assembled into 50S ribosomal subunits, and it can also catalyse the formation of 2′-O-methylribose in E.coli tRNA, although the modified nucleotide(s) has not yet been mapped (Bügl et al., 2000). In yeast, almost all types of RNA molecules contain 2′-O-methylnucleotides: tRNA, snRNA, small nucleolar RNA (snoRNA) and rRNA (Rozenski et al., 1999). Although transcription of these various RNAs takes place either in the nucleus (and the nucleolus) or in mitochondria, RNA maturation can be completed sequentially in every cellular compartment. For instance, several tRNA modifications occur after removal of the intron, probably after the tRNA has been transported to the cytoplasm (reviewed by Grosjean et al., 1997). Since the formation of methylnucleotides in eukaryotes takes place in different compartments, it could either be achieved by the same enzymes distributed in various compartments or catalysed by different enzymes located in the nucleus, the cytoplasm and the mitochondria. Remarkably, the yeast genome contains three open reading frames (ORFs) that exhibit significant sequence similarity to E.coli FtsJ/RrmJ: YBR061c (hereafter referred to as TRM7; see below), YGL136c (MRM2) and YCL054w (SPB1). Spb1p is a putative MTase from the yeast nucleolus that is involved in 60S ribosomal subunit synthesis (Pintard et al., 2000). In a separate report, we demonstrated that Mrm2p is a mitochondrial site-specific 2′-O-ribose MTase that catalyses the formation of Um2791 on the peptidyl transferase centre of the 21S rRNA (Pintard et al., 2002). Here, we report the results of experimental and bioinformatic analysis of Trm7p, which suggest that it is a new cytoplasmic 2′-O-RNA MTase. In yeast tRNAs, 2′-O-methylriboses have been found at positions 4, 18, 32, 34 and 44 (Grosjean et al., 1995; Sprinzl et al., 1998). Although certain modifications occur on the pre-tRNA in the nucleus, methylation at positions 18, 32, 34 and 44 appears to occur after the removal of the intron (reviewed by Grosjean et al., 1997). So far, Trm3p is the only putative yeast 2′-O-tRNA MTase that has been described. It is required for the site-specific formation of Gm at position 18 in the D-loop of intron-less tRNA (Cavaillé et al., 1999). Two of the remaining four 2′-O-methylnucleotides are located within the anticodon loop, at positions 32 and 34, in tRNALeu,Phe,Trp. Modification of position 34, the wobble nucleotide of the anticodon, has been reported to be important for translation efficiency and/or fidelity (Curran, 1998; Satoh et al., 2000). We report here that Trm7p is a yeast MTase that catalyses the formation of 2′-O-methylribose at positions 32 and 34 in the anticodon loop of various yeast tRNAs. We propose that the three proteins Trm7p, Spb1p and Mrm2p form a subclass of 2′-O-ribose MTases that have emerged from a single common ancestor and have co-evolved with their three different RNA substrates, located in the cytoplasm, the nucleolus and the mitochondria, respectively. Results Trm7p belongs to a family of putative MTases from Eukaryota that are structurally related to the 2′-O-rRNA MTase FtsJ/RrmJ from E.coli By searching the yeast genome, we identified three proteins exhibiting similarity to the bacterial 2′-O-rRNA MTase FtsJ/RrmJ (Caldas et al., 2000a, b): Spb1p (Pintard et al., 2000), Mrm2p (Pintard et al., 2002) and Ybr061c (referred to here as Trm7p; see below). We undertook the bioinformatic analysis to predict the structure of the three yeast proteins based on the data available for other MTases. The E.coli FtsJ/RrmJ sequence was used as a query in a PSI-BLAST search (Altschul et al., 1997) of the non-redundant amino acid sequence database at NCBI (http://www.ncbi.nlm.nih.gov/) with a stringent expectation value cut-off of 10−10. The search converged in the fifth iteration, yielding the alignment of highly conserved core segments. Sequences of 32 putative homologues of FtsJ/RrmJ were chosen and realigned to the PSI-BLAST core profile using Clustal_X (Thompson et al., 1997). A distance-based phylogeny was reconstructed according to the neighbour-joining method (Saitou and Nei, 1987), revealing three evolutionary lineages with representatives from yeast that were named the Trm7, Spb1 and Mrm2 subfamilies (Figure 1). Mrm2 groups together with the FtsJ/RrmJ family members from the Eubacteria and Archaea, suggesting that its ancestor has been brought into the eukaryotic cell via the mitochondrial endosymbiont. The topology of the tree suggests that a gene duplication event leading to Trm7 and Spb1 occurred in a common ancestor of the Eukaryota. Figure 1.Phylogenetic tree analysis reveals three putative orthologous lineages: the Trm7, Spb1 and Mrm2 families. The Mrm2 family is represented by Mrm2p from S.cerevisiae (P53123), FtsJ/RrmJ from E.coli (AAC76211) and other prokaryotic proteins [M.t., Methanobacterium thermoautotrophicum (G69103); M.j., Methanococcus jannaschii (Q58771); A.f., Archaeglobus fulgidus (O28228); R.p., Rickettsia prowazekii (Q9ZE00); N.m., Neisseria meningitidis (AAF41212); P.a., Pseudomonas aeruginosa (AAG08138); H.i., Haemophilus influenzae (P45162)] and eukaryotic proteins [A.t., Arabidopsis thaliana (CAB69851); C.e., Caenorhabditis elegans (O62251); D.m., Drosophila melanogaster (Q9VDT6); H.s., Homo sapiens (AAF22488); C.a., Candida albicans (unfinished sequence from the Stanford Genome Technology Center, SGTC, http://sequence-www.stanford.edu); S.p., Schizosaccharomyces pombe (P78860); and P.f., Plasmodium falciparum 3D7 (unfinished sequence from the P.falciparum Genome Project–PlasmoDB, http://www.plasmodb.org)]. The Spb1 family is represented by Spb1p from S.cerevisiae (CAA42391) and seven other members [A.t., A.thaliana (CAB69851); P.f., P.falciparum (unfinished sequence from the PlasmoDB); N.c., Neurospora crassa (CAB88626); S.p., S.pombe (T37754); C.a., C.albicans (unfinished sequence from the SGTC); C.e., C.elegans (AAF39868); and D.m., D.melanogaster (AAF48557)]. The Trm7 family is represented by Trm7p from S.cerevisiae (CAA85004) and eight other members [P.f., P.falciparum (unfinished sequence from the PlasmoDB); S.p., S.pombe (Z98980); C.a., C.albicans (unfinished sequence from the SGTC); H.s., H.sapiens (HSA005892); C.e., C.elegans (Q22031); A.t., A.thaliana (CAB69851); and D.m.1. and D.m.2., D.melanogaster (AAF55380 and AAF55857)]. Download figure Download PowerPoint The three-dimensional structures of the three putative MTases from yeast were homology modelled using Modeller (Sali and Blundell, 1993) based on the refined sequence alignment with FtsJ/RrmJ (Figure 2). The stereochemistry and pseudoenergetic features of the models passed all tests implemented in ProsaII (Z-scores ≤ −7.7; Sippl, 1993). Figure 2A shows the key elements of the predicted AdoMet- and ribose-binding sites of Trm7p, including the predicted catalytic tetrad of two basic and two acidic side chains (K28, D124, K164 and E199, each marked by an asterisk) conserved in many 2′-O-ribose MTases exhibiting the common 'MTase fold' (Bujnicki and Rychlewski, 2001). All three putative MTases from yeast and their orthologues (including E.coli FtsJ/RrmJ) exhibit striking conservation of the predicted active site and its neighbourhood, suggesting that their target must be similar. Figure 2.Three-dimensional structure prediction for Trm7p. (A) Alignment of the AdoMet-binding domain of the three yeast proteins (Trm7p, Spb1p and Mrm2p) with the E.coli protein FtsJ/RrmJ. Identical and chemically equivalent residues are highlighted in black and grey, respectively. Secondary structural elements are indicated above the alignment (α-helices as cylinders and β-strands as arrows). Conserved motifs have been labelled according to the nomenclature proposed by Posfai et al. (1988). Predicted interactions with AdoMet and the methylated nucleotide are designated by red arrowheads and green diamonds, respectively. The predicted catalytic tetrad K–D–K–E is labelled with purple stars. Two insertions in Mrm2p [I1 (58 amino acids) and I2 (21 amino acids)] have been omitted for the sake of clarity. (B) The stereogram of the Trm7p model in cartoon representation. The AdoMet moiety is shown in cyan, and the ribose and the phosphate group of the nucleotide to be methylated are shown in blue. The predicted binding and catalytic residues are shown in the wireframe representation, with colour coding analogous to that in (A). Download figure Download PowerPoint In the proposed model of interaction with an RNA substrate (Figure 2B), we docked the methylated ribose based on the superposition of the active sites of FtsJ/RrmJ and the yeast proteins with the coordinates of vaccinia mRNA cap-I 2′-O-MTase (Hodel et al., 1998). Although we cannot predict with confidence all specific interactions of the enzyme with the entire RNA molecule, we predict that the solvent-exposed side chains of residues S197 and R194 interact specifically with the phosphate group of the methylated nucleotide. Coordinates and additional structural representations are available (http://www.crbm.cnrs-mop.fr/~lapeyre/extpages/structure.html). Trm7p is able to bind tritiated AdoMet in vitro To challenge the model presented above, we tested the ability of a recombinant tagged Trm7p to bind AdoMet in vitro, as described previously for Spb1p (Pintard et al., 2000). Expression of Trm7′-ZZp (see Materials and methods) was able to complement the growth defect of a trm7Δ strain, thus demonstrating that the fusion protein was functional (Figure 3A). The level of expression of Trm7′-ZZp (Mr = 49 700) was compared with the level of the abundant ZZ-Nop1p by western blot analysis (Figure 3B). Signals obtained after serial dilutions indicate that Trm7p is ∼200 times less abundant in the cell than Nop1p, an observation that is in agreement with the codon adaptation index calculated for the two proteins (0.143 and 0.492, respectively; Sharp and Li, 1987). Figure 3.Trm7p binds tritiated AdoMet in vitro. (A) The doubling time was determined for various strains grown in YPD at 30°C. Open squares, wild-type strain (BMA64); open circles, trm7Δ strain (YBL4409); open triangles, trm7Δ transformed with a centromeric plasmid expressing the TRM7′-ZZ gene (YBL4494). (B) Relative expression of Trm7p. Cells expressing various tagged proteins were probed by western blotting using mouse IgG coupled to peroxidase (lower panel). Lane 1, Trm7′-ZZp (arrow on the left, strain YBL4494); lane 2, no tagged protein (BMA64); lane 3, ZZ-Nop1p (arrow on the right points to the major band, and a degradation product is detected below). Similar amounts of protein were loaded in each lane, as demonstrated using anti-Swi6p antibodies (upper panel). (C) Trm7′-ZZp was overexpressed from the GAL1-10 promoter (YBL4502), immunoprecipitated and incubated with 5 μCi of [3H]AdoMet before being UV crosslinked. Radiolabelled complexes were then denatured, separated by 10% SDS–PAGE and transferred to nitrocellulose. The membrane was first stained with amidoblack to localize the heavy and light chains of immunoglobulins used during the immunoprecipitation reaction (lanes 1 and 2). This membrane was then autoradiographed for 4 weeks at −70°C with an intensifying screen (lanes 3 and 4). Finally, the same membrane was analysed by western blotting to reveal Trm7′-ZZp (lanes 5 and 6). Lanes 1, 3 and 5, extracts prepared from the GAL::TRM7′-ZZ strain. Lanes 2, 4 and 6, extracts from an untagged strain as a control. Mr, molecular weight markers in thousands. Download figure Download PowerPoint Affinity-purified overexpressed recombinant Trm7′- ZZp was clearly capable of binding tritiated AdoMet in vitro (Figure 3C, lane 3). This binding strictly depends on UV irradiation, as is also the case for Spb1p (Pintard, 2000), thus demonstrating that labelling of the proteins was not due to alkylation by free methyl groups that could arise after degradation of AdoMet. Large amounts of immunoglobulins present in the reaction were not crosslinked to AdoMet under these conditions (Figure 3C, lane 1). Similarly, no signal was detected when bovine serum albumin or recombinant nucleolar Gar1p was used as control (data not shown), thus demonstrating the specificity of the reaction. This result strongly supports the view that Trm7p is a new MTase from Saccharomyces cerevisiae. Deletion of TRM7 impairs mRNA translation Sequence similarity existing between Trm7p and Spb1p led us to investigate whether Trm7p could also be a nucleolar protein. However, immunofluorescence studies performed with three different tagged proteins gave signals that were indistinguishable from the background noise. Intense signals were obtained only when TRM7 was overexpressed from the GAL1-10 promoter, revealing the protein throughout the cytoplasm (data not shown). Unlike Spb1p, Trm7p does not contain a nuclear localization signal, and PSORT software (Nakai and Kanehisa, 1992) predicts a cytoplasmic location for Trm7p (score = 0.650). Although we cannot definitely ascertain the location of the protein, since it was detectable only when overexpressed in the cell, our observations, combined with the PSORT prediction, support the view that Trm7 is a cytoplasmic protein. To understand better the role of Trm7p, we further analysed the phenotype of the trm7Δ strain. Incorporation of labelled methionine revealed that protein synthesis is reduced to 30% in the deleted strain, compared with an isogenic wild-type strain. This result was confirmed by the low level of polysomes detected in the mutant, compared with a wild-type strain, when extracts were fractionated on sucrose gradients (Figure 4A). We also noticed that the trm7Δ strain is highly sensitive to paromomycin, an antibiotic of the aminoglycoside family that impairs translation by increasing codon misreading in Prokaryota and Eukaryota (Figure 4B) (Chernoff et al., 1994). Taken together, these results suggests that Trm7p is a cytoplasmic protein that plays a role in mRNA translation. Figure 4.Translation is impaired in a trm7Δ strain. (A) Polysome profile analysis. Cellular extracts were fractionated onto 15–50% linear sucrose gradients. A continuous record of the absorbance at 254 nm for each gradient is presented, with the top of the gradient on the left. The arrows indicate the peaks for the 40S, 60S and 80S subunits. Extracts were prepared from the strains as indicated: WT, wild type (BMA64); trm7Δ (YBL4409). (B) The trm7Δ strain is sensitive to the translation inhibitor paromomycin. The wild-type strain (WT, BMA64) and the trm7Δ strain (YBL4409) were grown in YPD until mid-log phase; then 5 μl of 5-fold serial dilutions were spotted onto YPD plates containing (or not) 0.2 mg/ml paromomycin as indicated, and strains were grown for 3 days at 30°C. Download figure Download PowerPoint Trm7p is required for tRNA 2′-O-ribose methylation in vivo The close structural similarity existing between Trm7p and the E.coli rRNA MTase FtsJ/RrmJ suggests that Trm7p could be a new yeast 2′-O-RNA MTase. In vitro, FtsJ/RrmJ is also able to methylate tRNA (Bügl et al., 2000). This result, taken together with our observation that Trm7p is likely to be a cytoplasmic MTase that plays a role in mRNA translation, prompted us to test whether Trm7p was involved in 2′-O-ribose methylation of a nucleotide within a loop of yeast tRNA. Positions 32 and 34 in the anticodon loop represented good candidates that were tested further (Figure 5). Interestingly, when position 32 of a given tRNA is 2′-O-methylated, the wobble position 34 of the same tRNA is also 2′-O-methylated. To identify the modified nucleotides, we used the property that RNase T2 cleaves all phosphodiester linkages between nucleotides, except those that are 2′-O-methylated. Since the 3′-neighbouring nucleotides of each type of 2′-O-methyl derivative in the different isoacceptor tRNA are different, analysis of the dinucleotides produced by RNase T2 digestion reveals the identity of the modified nucleotide. The result of RNase T2 digestion of total tRNA extracted from various strains grown in the presence of inorganic [32P]orthophosphate, followed by two-dimensional thin-layer chromatography (TLC), is presented in Figure 6A–C. As expected, each tRNA digest revealed a complex pattern of radiolabelled spots, of which the four canonical 3′-monophosphate nucleosides Ap, Cp, Gp and Up are the most abundant. Spots corresponding to ribothymidine monophosphate (Tp), dihydrouridine monophosphate (Dp) and pseudouridine monophosphate (Ψp), which are present in all naturally occurring tRNA in yeast, are also clearly seen. Other modified nucleotides that are present only in certain tRNA yield spots that are less intense but still easily detected on the autoradiogram. Three spots (3, 6 and 8) of the wild-type strain (Figure 6A) are missing in the trm7Δ strain (indicated by arrows in Figure 6B). Spot 3 corresponds to GmAp (positions 34–35 in tRNAPhe), spot 6 corresponds to CmUp (positions 32–33 in tRNAPhe,Trp,Leu) and spot 8 corresponds to CmCp (positions 34–35 in tRNATrp). Two of these three radioactive spots are clearly visible in the trm7Δ strain transformed with the centromeric plasmid harbouring a synthetic TRM7′ gene (Figure 6C). Of note is the presence in all the chromatograms of the radioactive spots corresponding to CmAp (spot 1, from positions 4–5 in major tRNAGly), AmUp (spot 2, from positions 4–5 in tRNAHis), GmGp (spot 4, from positions 18–19 in several yeast tRNAs), UmGp (spot 5, from positions 44–45 in all tRNASer) and CmGp (spot 7, from positions 4–5 in major tRNAPro). The spot corresponding to the dinucleotide ncm5UmAp (positions 34–35 in minor tRNALeu) was not identified in this experiment, probably because it was in amounts too low to be detected and/or it co-migrates with another radioactive spot on the TLC plates. Figure 5.Position of the five 2′-O-methylnucleotides in the yeast cytoplasmic tRNA and its major domains. Positions in black indicate the site of methylation in tRNA containing 2′-O-methylnucleotide, and positions in grey correspond to its 3′-neighbouring nucleotide. The indicated dinucleotides correspond to those obtained after complete digestion of the tRNA by RNase T2. Download figure Download PowerPoint Figure 6.Position 32 of several tRNAs and position 34 of tRNAPhe and tRNATrp are not 2′-O-methylated in a trm7Δ strain. Autoradiograms of selected two-dimensional TLC of modified mono- and dinucleotides after RNase T2 digestion of various 32P-labelled tRNA recovered from cells that were grown in the presence of [32P]orthophosphate. Hydrolysate of total tRNA from (A) wild-type strain (BMA64α), (B) trm7Δ strain (YBL4409) and (C) trm7Δ strain transformed with a plasmid harbouring the TRM7′ gene (YBL4363) have been analysed with the N/R chromatographic solvent system. (D) A reference map indicating the location of the nucleotides of interest. Arrows point to the spots that disappear in the disrupted strain. Download figure Download PowerPoint Purification of wild-type and mutant recombinant Trm7 proteins As another means to demonstrate the catalytic activity of Trm7p, we mutated the AdoMet-binding domain of Trm7p to abolish cofactor binding by this enzyme. Based on the homology model of Trm7p and analysis of the FtsJ/RrmJ structure, D49 of Trm7p has been predicted to make an essential water-mediated interaction with AdoMet, the lack of which would interfere with cofactor binding. Therefore, a mutation was introduced into the TRM7 gene to change this aspartic residue into an alanine (→Trm7[D49A]). In vivo, a plasmid expressing the recombinant Trm7 wild-type protein tagged with six histidines (Trm7-H6p) was able to complement the growth defect of a trm7Δ strain, whereas expression of the mutant Trm7[D49A]-H6p in a trm7Δ strain did not restore wild-type growth (data not shown). Then, to assert unambiguously that the MTase activity was borne by Trm7p itself, the two recombinant and mutant proteins were affinity purified (Figure 7) and their tRNA MTase activity was tested in vitro. Few minor contaminants were detected by Coomassie Blue staining, the most abundant of which was also present in a fraction prepared from a trm7Δ strain expressing no recombinant His-tagged protein (Figure 7, lane 7). Figure 7.Purification of the His-tagged wild-type and mutant recombinant Trm7 proteins. Affinity-purified wild-type and D49A mutant Trm7p were analysed by 10% SDS–PAGE. To assess the purity of the proteins, various amounts were loaded (∼0.5, 1 and 2 μg, from left to right, as indicated). Lanes 1–3, wild-type Trm7-H6p; lanes 4–6, D49A mutant Trm7-H6p; lane 7, control (C) from an untagged strain. Mr, molecular weight markers in thousands. Download figure Download PowerPoint Trm7p is required for tRNA 2′-O-ribose methylation in vitro To test in vitro for AdoMet-dependent MTase activity at positions 32 and 34, a synthetic tRNA substrate was prepared. Intron-less yeast tRNAPhe was transcribed in vitro using [α-32P]GTP as a radioactive precursor. This permits, using the same transcript, the detection of the presence of either CmU32p after RNase T2 digestion or 32pGm after nuclease P1 digestion. When this substrate was incubated with a S10 cellular extract prepared from a wild-type strain, enzymatic formation of both Cm32 and Gm34 was detected, although a small but reproducible lag phase in the formation of Gm34 was visible (Figure 8, left part). Formation of Cm32 and Gm34 was completely abolished in an S10 extract from a trm7Δ yeast strain (Figure 8B and D). Similar results were obtained with an intron-less yeast tRNATrp transcript (data not shown). Several other radioactive spots were generated by the S10 extract from the wild-type strain, which were also present in the trm7Δ strain extract: Dp from position 17, in addition to the expected CmUp after T2 RNase digestion; and pm2G, pm22G, pm1G and pm7G from positions 10, 26, 37 and 46, respectively, in addition to pGm after nuclease P1 digestion (Figure 8A and C). This result demonstrates that only the formation of Cm32 and Gm34 is abolished in the trm7Δ strain. Moreover, enzymatic formation of Cm32 and Gm34 was restored when the S10 extract was prepared from a trm7Δ strain transformed with a centromeric plasmid expressing the TRM7 gene (data not shown). Figure 8.Affinity-purified Trm7p is able to methylate positions 32 and 34 of tRNAPhe in vitro. Upper part: autoradiograms of selected two- dimensional TLC of modified mono- and dinucleotides after RNase T2 (A, B, E and F) or nuclease P1 (C, D, G and H) digestion of synthetic intron-less [α-32P]GTP-labelled tRNAPhe initially incubated either with S10 cell extracts or with purified recombinant proteins. Left four panels: tRNA incubated for 2 h at 30°C with 2 mg/ml protein from an S10 cell extract prepared from a wild-type strain (BMA64α) (A and C) or from a trm7Δ strain (YBL4409) (B and D). Right four panels: tRNA incubated as above at 30°C with 10 μg/ml purified Trm7-H6p (E and G) or with D49A mutant Trm7-H6p (F and H). Lower part: time-course formation of Cm32 (detected as CmU) and Gm34 on synthetic intron-less [α-32P]GTP-labelled tRNAPhe incubated at 30°C either with S10 extracts (left panel) or with recombinant proteins (right panel). Molar ratios of methylated nucleotides versus substrate tRNA plotted as a function of incubation time in minutes (methylnucleotides/tRNA). Left panel: S10 extracts prepared from a wild-type yeast (filled circles, CmU; filled squares, Gm) or from a trm7Δ mutant strain that does not give rise to any detectable modification (open circles and open squares, respectively). Right panel: purified recombinant wild-type Trm7-H6p (filled circles, CmU; filled squares, Gm) or with purified mutant Trm7[D49A]-H6p (open circles and open squares, respectively). Download figure Download PowerPoint The same synthetic tRNA substrate was then incubated with the recombinant proteins, either the wild-type or the D49A mutant Trm7p, and analysed as above for the presence of modified nucleotides. The wild-type protein was clearly capable of driving the formation of both Cm32 and Gm34, although the rate of Cm32 formation was ∼10 times faster than the rate of Gm34 formation in intron-less tRNAPhe, and a slight but reproducible lag phase exists for the enzymatic