INTRODUCTION

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The modified nucleoside pseudouridine (5-(-D-ribofuranosyl)uracil, abbreviated as ),¹ is found very frequently in all kinds of RNA from eubacteria, archaea, and eukaryotes (1). It is present in all transfer RNAs (2), large and small subunits of ribosomal RNA (3, 4), most small nuclear RNAs (5, 6), and selected small nucleolar RNAs (7).

The numerous  residues in RNA are produced by a family of enzymes (pseudouridine synthases, RNA: synthases). These enzymes act on specific uridine residues of the RNA molecules, but still very little is known about the number of these enzymes in a given cell as well as their mechanism and their RNA recognition mode.

In yeast tRNA, formation of pseudouridines at positions 13, 32, and 55 is catalyzed by three distinct enzymes (8), whereas in the same yeast cell one single enzyme (Pus1p) is responsible for  formation at positions 27, 34, and 36 (9). Several distinct pseudouridine synthase activities acting on eukaryotic small nuclear RNAs were also detected in crude cell extracts (5); however, none of these enzymes has been identified so far. From recent works on rRNA maturation, it appears that most (if not all) of the  in eukaryotic rRNA is probably synthesized by a single (or at least very few) rRNA: synthase(s); in this latter case the enzyme(s) is(are) guided to the different target uridines within the rRNA by a huge family of diverse small nucleolar ribonucleoproteins present in the nucleolus (10, 11; for review, see Ref. 12).

The first RNA:pseudouridine synthase that was purified and fully characterized came from Salmonella typhimurium (13) and later from Escherichia coli (14). It corresponds to a tRNA: synthase (PSU-I, previously called hisT gene product, recently renamed to truA). It catalyzes the formation of ₃₈, ₃₉, and/or ₄₀ in several cellular tRNAs. Only the gene for the E. coli enzyme (PSU-I) was cloned and sequenced (15). The E. coli PSU-Ip is a monomer with a molecular mass of 31 kDa (270 amino acids), whereas PSU-Ip from S. typhimurium has a subunit size of about 50 kDa and dimerizes in the presence of tRNA substrate (13). A higher eukaryotic homolog of PSU-I from calf thymus was highly enriched after five chromatographic steps (16); however, a homogeneous preparation was not obtained. All of these enzymes have similar properties: they do not require any cofactor, they catalyze the  formation at contiguous sites (region specificity), and they demonstrate similar kinetic properties (13, 14, 16).

Recently three other E. coli RNA: synthases were identified and their corresponding genes cloned: truB, which is site-specific for  at position 55 in tRNA (17); rluA, specific for  at position 32 in tRNA and at position 746 in 23 S rRNA (dual specificity; Ref. 18); and rsuA, specific for U at position 516 in 16 S rRNA associated with ribosomal proteins within a ribosomal ribonucleoprotein complex (19).

The genes for two other tRNA:pseudouridine synthases (Pus1p and Pus2p) from yeast were also identified, one of them (PUS1) was cloned and overexpressed in E. coli (9). The recombinant yeast Pus1p was shown to be specific for  at position 27 in several yeast tRNAs and for  at positions 34 and 36 in the intron-containing pre-tRNA^Ile (9; for review, see Ref. 20). This enzyme has a molecular mass of 62 kDa and was shown to be located essentially in the yeast nucleus (9). The target RNA and position(s) of uridine to be modified by Pus2p (a 42-kDa protein) have not been determined yet.

Another yeast protein has been reported to be homologous to E. coli and S. typhimurium pseudouridine synthases PSU-I (hisT gene product, truAp) (21). This protein (called Deg1 because disruption of the gene causes depressed growth; SacchDB accession number YFL001W, SwissProt P31115) also displays a significant homology to the yeast Pus1p (9). Based on this sequence homology with PSU-I, it was suggested (21) that Deg1 protein may possess the corresponding activity of hisT gene product, but this plausible hypothesis was never tested experimentally. Here we present experimental evidence that yeast protein Deg1 is indeed a new distinct yeast tRNA: synthase (Pus3p) with a slightly different specificity toward tRNA compared with E. coli enzyme truA since it catalyzes the formation of only ₃₈ and ₃₉ (not ₄₀). The structural substrate requirements and the intracellular localization of Pus3p are also analyzed.

	EXPERIMENTAL PROCEDURES

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Chemicals, Enzymes, and Materials-- -³²P-Radiolabeled nucleotide triphosphates (400 Ci/mmol) were from Amersham (U. K.). Tris, dithioerythritol, dithiothreitol, nucleoside triphosphates, Penicillium citricum nuclease P1, Aspergillus oryzae RNase T2, and phenylmethylsulfonyl fluoride were from Sigma. Diisopropyl fluorophosphate and CMCT were from Aldrich. Bacteriophage T7 RNA polymerase, restriction enzymes, and isopropyl-1-thio--D-galactopyranoside were from from MBI Fermentas (Vilnius, Lithuania). RNasin and avian myeloblastosis virus reverse transcriptase were from Promega. Synthetic oligodeoxynucleotides used as primers for reverse transcription were purchased from MWG-Biotech (Germany) and used without further purification. Thin layer cellulose plates were from Schleicher & Schuell (Germany), and all other chemicals were from Merck Biochemicals (Germany).

Plasmids and Transcription of tRNA Genes-- The plasmids carrying the synthetic genes of tRNAs used in this work were described previously tRNA^Asp (GUC) (22), pre-tRNA^Ile (UAU) and its mutant (anticodon UUA) (23), pre-tRNA^Tyr (mutant UUA) (24), and E. coli tRNA^Ser (GGA) (25). Plasmids with cloned genes of yeast tRNA^Phe (GAA) and its mutants PheY54 (p67YF1, mutation C56G) and PheY55 (p67YF2, mutations G19C and C56G) have been described (26). Plasmids carrying yeast tRNA^His (GUG), yeast tRNA^Ser (IGA), and mutant yeast tRNA^Val (harboring anticodon CAU instead of UAC) were gifts, respectively, of Dr. J. Rudinger (IBMC, Strasbourg), Dr. H. Himeno (Tokyo University, Japan), and Dr. F. Fasiolo (IBMC). The synthetic gene for yeast tRNA^Ala (anticodon IGC) was constructed by ligation of sets of complementary oligonucleotides as described (22).

Yeast Strains, Media, and Microbiological Techniques-- The wild type yeast strain used in this study was RS453 (/a, ade2/ade2, leu2/leu2, ura3/ura3, his3/his3, trp1/trp1), and all mutant strains containing disrupted genes were derived from this one. Yeast cells were grown on minimal SDC and rich YPD medium (37), and sporulation of diploid cells on YPA plates and tetrad analysis were performed according to Ref. 29. Minimal SDC medium/plates were supplemented by all amino acids and nutrients except the ones used for the selection or, if indicated, contained 5-fluoroorotic acid (CSM medium, BIO 101, La Jolla). Genetic manipulations were performed as described (30).

Cloning and Expression of Yeast Deg1p in E. coli-- Preparation of the construct for expression of Deg1 in yeast was done as follows. The DEG1 gene was amplified by PCR from total yeast genomic DNA using two primers that created an XbaI restriction site in the 5-untranslated region of the gene 151 nucleotides upstream of the start codon (ttttttctagAATCAATGGGCTCAGCTC, complementary sequence in capital letters) and an SalI restriction site (tttttgtcgacAAGAAATATAGTCTTCAAGG) in the 3-untranslated region of the gene 50 nucleotides downstream of the stop codon. This allowed cloning of the gene into a pRS315 vector previously cut with XbaI/SalI. This construct could complement the slow growing phenotype when transformed in the deg1 haploid cells.

Gene Disruption of DEG1-- Disruption of the DEG1 gene was done by the one-step gene replacement method (31). In this study, the DEG1 gene was disrupted by inserting a BamHI fragment 0.9 kilobases long and containing the HIS3 gene into the BamHI site of the DEG1-ORF cloned in the pET8c vector. The disrupted gene was excised and the linear fragments used to transform the diploid strain RS453. HIS⁺ transformants with the correct integration of the interrupted gene at the DEG1 locus were verified by PCR analysis (data not shown). Correct integrants were sporulated, and tetrads were dissected. A 4:0 segregation for viability and a 2:2 segregation for the HIS marker were found for the DEG1 gene disruption showing that this gene is not essential for cell growth. However, all spores carrying a disrupted DEG1 gene grew slower giving rise to small colonies as reported previously (21).

Construction of Doubly Disrupted Mutants-- To construct a haploid yeast strain in which the disrupted PUS1 and DEG1 genes are combined, a PUS1 mutant harboring pURA3-PUS1 was mated to the mutant DEG1. The resulting heterozygous diploids were sporulated, and tetrad analysis was performed. For complete tetrads in which the HIS⁺/his genotype segregates 2:2, one can predict that the two HIS⁺ progeny are deg1::HIS3/pus1::HIS3. A complete tetrad showing this segregation pattern was analyzed in greater detail for the segregation of the HIS3 and URA3 markers by plating cells on SDC-his and SDC-ura plates, respectively. The HIS⁺ progeny deg1::HIS3/pus1:HIS3 also contained the plasmid pURA3-PUS1; we could shuffle out this plasmid and test whether the double mutant pus1/deg1 gives synthetic lethality by plating this strain on 5-fluoroorotic acid containing plates at 30 °C. The los1/deg1 strain was constructed in a similar way.

Construction of the Deg1p Fusion Protein Carrying Protein A or Green Fluorescent Protein (GFP) as a Tag-- Epitope tagging of Deg1p was done by fusing two IgG binding units from Staphyloccus aureus protein A to the N-terminal end of Deg1p. For this gene fusion, a new PstI restriction site was generated at the ATG codon of DEG1 by PCR-mediated mutagenesis, and the ORF was subcloned into the plasmid pRS315 in-frame with the two IgG binding units under the control of the NOP1 promoter (P_Nop1-ProtA cassette; see Ref. 32), creating the plasmid pNOP-ProtA-Deg1. Affinity purification of the ProtA fusion protein was done as described previously (32). Tagging with GFP was done in a similar way, but the IgG binding units were replaced by the ORF for GFP (P_Nop1-GFP cassette),² creating the plasmid pNOP-GFP-Deg1. The GFP used is a S65T/V163A variant exhibiting enhanced fluorescence properties (33, 34).

Cellular Localization of DEG1 Gene Product-- Intracellular localization of the ProtA-Deg1p fusion protein was performed by indirect immunofluorescent microscopy as described in Ref. 9 using as first antibody rabbit anti-protein A (Sigma) and as second antibody Cy^TM3-conjugated AffiniPure donkey anti-rabbit IgG (Dianova). GFP-Deg1p was observed in living cells by direct fluorescent microscopy.

Pseudouridine Formation Assay in Vitro-- Preparation of S100 extract from yeast as well as from E. coli was made as described elsewhere (25, 28). The activity of yeast extracts and purified yeast enzyme fractions was tested at 30 °C; 37 °C was used for testing E. coli extracts. The incubation mixture contained 100 mM Tris-HCl, pH 8.0, 100 mM ammonium acetate, 5 mM MgCl₂, 2 mM dithiothreitol, 0.1 mM EDTA, and 1-2 fmol of ³²P-radiolabeled T7 runoff transcripts as substrate. After incubation, the pseudouridine content in the radiolabeled transcripts was analyzed as described previously (27, 28). In brief, the RNA was first extracted with phenol-chloroform, precipitated in ethanol, and then hydrolyzed completely to 3-nucleotide monophosphates by RNase T2. Each hydrolysate was chromatographed on two-dimensional thin layer chromatography plates, and the radioactivity in the MP and UMP spots was evaluated after exposing the thin layer chromatography plates with a PhosphorImager screen. Taking into account the relative number of MP and UMP in the tRNA molecule, the relative amount of  over tRNA molecule (expressed in mol/mol of tRNA) can be evaluated. The accuracy of this method was found to be about ± 0.05 mol of /mol of tRNA.

Identification and Localization of Naturally Occurring  Residues in tRNAs-- Localization of pseudouridine residues in tRNA was performed as described (35, 36) with the following modifications. 10 µg of total tRNA extracted from wild type or mutant yeast strain was treated by 0.17 M CMCT in Bicine-urea buffer, pH 7.5, for 15 min at 42 °C. Reverse transcription was done at 42 °C for 45 min using about 1 µg of CMCT-modified cytoplasmic tRNA or 3 µg of CMCT-modified mitochondrial tRNA and 1-2 pmol of 5-³²P-labeled synthetic oligodeoxynucleotide primer. Reverse transcription products were separated on 15% denaturing polyacrylamide gel. The oligodeoxynucleotide primers were chosen to be complementary to the 18 nucleotides at the 3-end of cytoplasmic tRNA^Gly and mitochondrial tRNA^Arg, respectively, because both of these tRNAs have a low content of modified nucleotides downstream from the pseudouridylation sites.

Miscellaneous-- Isolation of total yeast DNA was done essentially as described in Ref. 37. DNA and plasmid manipulations (restriction analysis, end filling reactions, ligations, PCR amplifications, DNA fragment recovery, and small scale and large scale plasmid preparations) were done essentially according to standard procedures (38). The nucleotide sequence of tRNA inserts in the various plasmids used was verified systematically by the dideoxy sequencing technique. Evaluation of the protein concentration was done according to Bradford (39), and Western blotting was performed as described in Ref. 9.

	RESULTS

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Yeast Protein Deg1 Displays Significant Sequence Homology with E. coli truA and Yeast Pus1-- The accessibility of whole genome sequencing data for several prokaryotic and eukaryotic organisms allows the systematic screening for the corresponding enzymes based on sequence homology. BLAST search, using the hisT(truA) gene of E. coli (coding for tRNA:_38-40 synthase; see Ref. 15) as a query sequence, allows detection of several homologous proteins in the GenBank. Partial sequence alignment of hisT(truA)-like proteins of different origin is presented on Fig. 1. These are mostly putative pseudouridine synthases I (PSU-I) from several bacteria and lower eukaryotes. Higher eukaryotes are presented by the partial nucleotide sequence of human (zn81c07.s1) Deg1-like protein. All of these putative proteins display significant sequence homology with E. coli truA (BLAST p value is less than 0.001). All proteins share common signature blocks of amino acid, which suggests the same or similar function in the cellular metabolism. Here it is noteworthy to remember that the yeast PUS1 gene also shares high homology with the truA(hisT) gene, but the corresponding RNA: synthase has a different specificity and properties (9). Also, as already noted by Koonin (40), this group of hisT(truA)-like enzymes is rather distant from the pseudouridine synthases identified so far, specific, respectively, for ₅₅ (E. coli truB-like), for ₃₂ in tRNA and ₇₄₆ in 23 S rRNA (E. coli rluA-like), and for ₅₁₆ in E. coli 18 S rRNA. From the above sequence homology one can expect that protein Deg1 has enzymatic activity similar to that of E. coli truA.

View larger version (99K): [in this window] [in a new window]   Fig. 1.   Conserved sequence motifs in a family of truA-like proteins. Putative pseudouridine synthases I (38/39/40) from Haemophilus influenzae (U32837), Mycoplasma genitalium (U39695), B. burgdorferi (Y09141), Bacillus subtilis (D64126), Synechocystis sp. (D90905), Mycoplasma pneumoniae (U34795, putative 28-kDa protein), Bacillus sp. (M84963, ORF 5-adjacent to endo-1,4--glucanase gene), Methanococcus jannaschii (U67608), and Caenorhabditis elegans (Q09524) are aligned with tRNA:38/39/40 synthase from E. coli (TRUA_ECOLI, P07649) and Saccharomyces cerevisiae (Deg1, P31115). Higher eukaryotes are presented by the partial nucleotide sequence of human (zn81c07.s1) Deg1-like protein. Yeast pseudouridine synthases Pus1 and Pus2 are presented on the bottom. The number of amino acid residues between blocks and from the protein termini are indicated by numbers. The consensus line shows the domains of amino acids that are conserved in the majority of sequences. Universally or highly conserved amino acid residues are shaded. U in the consensus line indicates a hydrophobic residue. The invariant aspartic acid residue (D) in block II is indicated by dot. The accession numbers of each sequence and their respective kingdom (P for prokaryotes, A for archaea, E for eukaryotes) are given at the end of each line. The abbreviated names of organisms corresponds to that in SwissProt.

Disruption of Deg1 Gene Results in the Disappearance of tRNA:_38/39 Enzymatic Activity in the Yeast Cell Extract-- To test the function of yeast Deg1 protein, we performed one-step gene disruption of the corresponding gene in yeast. The resulting yeast strain remains viable, but the deg1 mutant grows slower compared with parent wild type strain cells as was reported previously (21). Furthermore, we observed that the slow growth phenotype is particularly strong at 37 °C (Fig. 2). This thermosensitive phenotype and growth defect of the deg1 strain could be fully complemented by expressing the cloned DEG1 gene or the fusion proteins ProtA-Deg1p and GFP-Deg1 (Fig. 2 and below).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Disruption of DEG1 leads to slow growth and a temperature-sensitive phenotype. Serial dilutions of wild type strain (RS453), deg1 strain, and deg1 strain transformed by plasmids encoding for Deg1p or N-terminally tagged forms were spotted on YPD plates and incubated for 3 days at the indicated temperatures.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Time courses of 38 and 39 formation in the extract of wild type (filled symbols) and DEG1-disrupted yeast strains (open symbols). Pseudouridine formation was measured using as substrates the transcript of yeast tRNAAla for 38 formation (squares) and tRNAHis for 39 formation (circles).

_38/39 Synthase Activity Resides in the Deg1 Protein-- To show that Deg1p is solely responsible for the modifications missing in the DEG1-disrupted strain, the recombinant protein was expressed in E. coli and its activity tested in vitro. The results show that the activity of tRNA:_38/39 synthase is readily detected in the extract of E. coli expressing His₆-Deg1 and also upon the fractionation of the induced extract on Ni²⁺-nitrilotriacetic acid-agarose. Activity of the expressed yeast pseudouridine synthase was retained considerably by metal affinity column, and activity toward ₃₈ in yeast tRNA^Ala was detected only in the case of induced E. coli extract. Similar results were obtained using purified by S-Sepharose FF, hydroxyapatite, and Ni²⁺-nitrilotriacetic acid-agarose column recombinant Deg1p (more than 95% purity), which efficiently catalyzes the pseudouridine formation in the transcripts of yeast tRNA^Phe (₃₉) and tRNA^Ala (₃₈) (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Panel A, affinity purification of ProA-Deg1p by IgG-Sepharose chromatography. Fractions of the soluble total cell extracts (S), the column flow-through (FT), the pH 5 wash (W), and the pH 3.4 eluate (E) were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie staining (left panel) or immunoblotting with antibodies that react with the protein A moiety of the fusion protein (right panel). Panel B, 39 and 38 formation in mutant yeast tRNAPhe (PheY55) and yeast tRNAAla using the suspension of IgG-Sepharose with bound ProtA-Deg1 fusion as the source of enzymatic activity. Time points at 30 and 60 min are presented for 39 formation by two independent preparations of ProtA-Deg1 (shown as filled and shaded bars). Only the 60-min point was measured for 38 formation.

Specificity of Yeast and E. coli tRNA:_38-40 Synthases Is Different-- We compared the specificity of the yeast and E. coli enzymes using the transcripts of various yeast and E. coli tRNAs. Fig. 5 shows the anticodon stem-loop regions of the naturally occurring tRNAs, the transcripts of which were tested for the formation of ₃₈, ₃₉, and ₄₀. As shown in Fig. 6A (see also Table II), the transcript of yeast tRNA^His (anticodon GUG) is modified efficiently to pseudouridine at position 39 upon the incubation in yeast or E. coli extract.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Anticodon stem-loops of naturally occurring yeast and E. coli tRNAs. In this study we used as substrates the corresponding runoff transcripts, lacking all modified nucleotides.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Panel A, time courses of 38, 39, and 40 formation in the transcripts of yeast tRNAAla (triangles), yeast tRNAHis (squares), and E. coli tRNASer (circles) incubated, respectively, with yeast (open symbols, solid lines) and E. coli extracts (filled symbols, dashed lines). Panel B, time courses of pseudouridine 39 formation in a wild type and mutants of yeast tRNAPhe(GAA). Wild type yeast extract was used as the source of enzyme. Wild type tRNAPhe () and mutants PheY54 () and PheY55 ().

Deg1p Activity Is Sensitive to tRNA Three-dimensional Structure-- Yeast tRNA^Phe (anticodon GAA) is an excellent experimental model to study the influence of overall three-dimensional tRNA structure on the modification reaction. This tRNA naturally contains 14 modified nucleosides, among them two pseudouridine residues (at positions 39 and 55). The set of tRNA^Phe mutants with well defined disruptions in tertiary interactions was also available (26). As shown in Fig. 6B and Table II, almost 1.7 mol of /mol of tRNA^Phe transcript was formed upon the incubation with the yeast extract.

Intracellular Location of Deg1p-- The subcellular localization of ProtA-tagged Deg1p in yeast cells was analyzed by indirect immunofluorescence microscopy using tag-specific antibodies and by direct fluorescence microscopy using a GFP-tagged version of Deg1p which could also complement the deg1 strain. In both cases a specific intranuclear signal could be detected, but the cytoplasm was also diffusely stained (Fig. 7). These results show that Deg1p resides in both the nucleus and the cytoplasm.

View larger version (78K): [in this window] [in a new window]   Fig. 7.   Cellular localization of Deg1. ProtA-Deg1p was localized by indirect immunofluorescent microscopy. The same sample was also stained for DNA. GFP-Deg1p was observed in living cells by direct immunofluorescent microscopy. The same cells are also shown by Nomarski optics.

Pseudouridine Residues ₃₈ and ₃₉ Are Absent in tRNA from Deg1 Strain-- The cellular localization of Deg1p studied by immunofluorescence techniques reveals the presence of nuclear and cytoplasmic pools of the protein but leaves open the question about its presence in yeast mitochondria. To answer this question we performed the analysis of pseudouridine residues present (or absent) in tRNAs extracted from wild type and DEG1-disrupted strain. Chemical mapping of pseudouridines was done on total tRNA fraction by CMCT-reverse transcription technique, as described previously (35). The synthetic oligonucleotides, complementary to the last 18-20 3-nucleotides in tRNA, were used for primer extension analysis using unmodified and CMCT-treated tRNA. The results of reverse transcription for cytoplasmic tRNA^Gly (anticodon GCC) and mitochondrial tRNA^Arg (anticodon ACG) extracted from wild type and mutant strains are presented in Fig. 8, A and B. The strong reverse transcription stops corresponding to ₃₈ in cytoplasmic tRNA^Gly and ₃₉ in mitochondrial tRNA^Arg are totally absent in the mutant DEG1-disrupted strain (indicated by arrows). The formation of other pseudouridines naturally present in these two tRNAs (₃₂ and ₅₅ in cytoplasmic and mitochondrial tRNA respectively) is not affected by DEG1 gene disruption. These results confirm the absolute requirement of Deg1 protein for modification of the pool of cytoplasmic tRNAs and reveal that the product of the same gene DEG1 participates also in pseudouridine formation in mitochondrial tRNAs.

View larger version (108K): [in this window] [in a new window]   Fig. 8.   Chemical mapping of pseudouridine residues in cytoplasmic tRNAGly (panel A) and mitochondrial tRNAArg (panel B) from wild type and DEG1-disrupted yeast strains. Autoradiographies of reverse transcription products in 15% polyacrylamide and 8 M urea gels are shown. The strong stops (shown by arrows) in reverse transcription of tRNA correspond to pseudouridine residues. The corresponding tRNA sequencing is shown on the left of each gel. Control tRNA samples and samples of tRNA treated by CMCT followed by sodium bicarbonate hydrolysis are presented side by side.

Deg1p Is Not Linked Genetically to Pus1p or Los1p-- We have shown previously that Pus1p interacts genetically with the nuclear pore-associated proteins Nsp1p and Los1p (9), suggesting that tRNA modification may be linked to tRNA nuclear export. To test this possibility for Deg1p, we combined the deg1 disruption with the los1 or pus1 disruption by mating the corresponding strains and performing tetrad analysis. The double-disrupted haploid strains los1/deg1 and pus1/deg1 were viable and did not exhibit any further growth defect than the single-disrupted deg1 strain (data not shown). Therefore, the functional interaction with the nuclear pore complex appears to be specific for Pus1p and does not occur in the case of Deg1p, suggesting that modification only at particular sites is important for the tRNA transport process. Furthermore, the viability of the pus1/deg1 strain shows that yeast cells can tolerate the absence of  modification in positions 27, 34, 36, 38, and 39. Thus, despite the different specificity of Pus1p and Pus3p there is no obvious synergism between them.

	DISCUSSION

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Yeast Protein Deg1 Is tRNA:_38/39 Synthase-- Direct evidence that the yeast Deg1 protein is a tRNA:pseudouridine synthase comes from two types of experiments: by detecting the corresponding enzymatic activity in S100 extracts of a transformed E. coli strain harboring the yeast DEG1 gene and by measuring the enzymatic activity of a ProtA-Deg1 fusion protein purified from a deg1 yeast strain. In both cases, the protein catalyzes in vitro formation of pseudouridines at position 38 or 39 of several tRNA transcripts. Additional evidence came also from identification of the lacking pseudouridines at position 38 or 39 in cytoplasmic tRNA^Gly and in mitochondrial tRNA^Arg present in DEG1-disrupted yeast strain. As predicted previously (21), Deg1p is indeed the yeast homolog of E. coli tRNA: synthase I (PSU-I, also called truA, initially discovered as hisT gene product). Therefore, after Pus1 and Pus2 (9), this is the third tRNA: synthase (PseudoUridine Synthase, Pus3) so far characterized in yeast.

Yeast Pus3 and E. coli truA Display Different Substrate Specificity-- Testing several tRNA substrates allowed us to reveal subtle differences in substrate specificity between yeast Deg1p and E. coli truAp. Indeed, although both enzymes display rather good cross-reactivity toward uridine 39 in two different tRNA transcripts (tRNA^Phe and tRNA^His), the situation for ₃₈ and ₄₀ formation was different. E. coli truA modified U₄₀ fairly well in both E. coli tRNA^Ser and yeast tRNA^Asp, whereas the yeast Deg1p did not modify U₄₀ at all in the same tRNA transcripts. This observation fits well with the fact that U₄₀ in naturally occurring yeast tRNA^Asp is not modified into , but in E. coli all U₄₀-containing tRNAs bear ₄₀ (42). This absence of ₄₀ is valid only for tRNAs from fungi and does not apply for tRNAs from higher eukaryotes, where few cases of ₄₀-containing tRNAs were found (42). However, the above observation for U/₄₀ relationship in yeast tRNAs is valid for both yeast cytoplasmic and the mitochondrial tRNAs, as in cytoplasm, several naturally occurring yeast mitochondrial tRNAs contain ₃₈ or ₃₉ but not ₄₀. This is consistent with the fact that the same gene product modifies all tRNA substrates in the different cellular compartments in yeast (nucleus, cytoplasm, and mitochondria; see below).

A Single Nuclear Gene DEG1 Provides the Enzyme for Three Cellular Compartments-- Immunochemical studies demonstrate that Deg1 protein is mostly located in the nucleus, but a significant part of it is also found in the yeast cytoplasm. Moreover, the analysis of the pseudouridine tRNA modification pattern in the DEG1-disrupted strain demonstrates clearly that a single gene DEG1 is responsible for the enzymatic formation of _38/39 in both cytoplasmic and mitochondrial tRNAs.

Deg1p Belongs to a truA-like Family That Is Distinct from All Other RNA: Synthases Sequenced So Far-- Comparison of the amino acid sequences of 12 truA-like proteins identified so far from different organisms reveals the presence of highly conserved residues (shown in gray in Fig. 1) within six blocks that are common to all proteins of the family. Interestingly, almost the same signature is present in Pus1p and Pus2p but is not found in any other protein of the whole protein data bank (SwissProt and GenBank). Only the sequence GRTDXGVHXG (block II in Fig. 1), bearing a conserved aspartic acid residue (indicated by a dot in Fig. 1), is similar to amino acid sequence GXRDXXXG (also referred as block II by Koonin (40)) present in all other RNA:pseudouridine synthases. As already proposed by Koonin, this universally conserved aspartic acid residue may be implicated in the enzymatic catalysis. Recent cross-linking studies and site-directed mutagenesis performed on E. coli tRNA pseudouridine synthase I (prokaryotic homolog of Pus3) clearly demonstrate that Asp-60 residue is essential for enzymatic activity (46). Successful crystallization and resolution of the structure of E. coli tRNA pseudouridine synthase I have also been reported (47), which should shed light on the catalytic mechanism of the uridine isomerization.

Disruption of the DEG1 Gene Influences Yeast Cell Growth-- In yeast, gene DEG1 was discovered incidentally by transcriptional analysis of the centromere region of yeast chromosome VI. This gene is located very close to the centromere and is expressed at only a low level. Its disruption, although not lethal, causes a pronounced slow growth phenotype (DEpressed Growth) (21). In this work, we show that this slow growth phenotype, which is much more pronounced at 37 °C, is entirely the result of the absence of Deg1 protein as it can be complemented by the corresponding gene or by clones expressing N-terminally tagged proteins. The absence of the Deg1p correlates with the absence of  at positions 38 and 39 of tRNA.