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Identification of Two tRNA Thiolation Genes Required for Cell Growth at Extremely High Temperatures

2006; Elsevier BV; Volume: 281; Issue: 20 Linguagem: Inglês

10.1074/jbc.m511675200

1083-351X

Naoki Shigi, Yuriko Sakaguchi, Tsutomu Suzuki, Kimitsuna Watanabe,

Biochemical and Molecular Research

Thermostability of tRNA in thermophilic bacteria is effected by post-transcriptional modifications, such as 2-thioribothymidine (s2T) at position 54. Using a proteomics approach, we identified two genes (ttuA and ttuB; tRNA-two-thiouridine) that are essential for the synthesis of s2T in Thermus thermophilus. Mutation of either gene completely abolishes thio-modification of s2T, and these mutants exhibit a temperature-sensitive phenotype. These results suggest that bacterial growth at higher temperatures is achieved through the thermal stabilization of tRNA by a 2-thiolation modification. TtuA (TTC0106) is possibly an ATPase possessing a P-loop motif. TtuB (TTC0105) is a putative thio-carrier protein that exhibits significant sequence homology with ThiS of the thiamine synthesis pathway. Both TtuA and TtuB are required for in vitro s2T formation in the presence of cysteine and ATP. The addition of cysteine desulfurases such as IscS (TTC0087) or SufS (TTC1373) enhances the sulfur transfer reaction in vitro. Thermostability of tRNA in thermophilic bacteria is effected by post-transcriptional modifications, such as 2-thioribothymidine (s2T) at position 54. Using a proteomics approach, we identified two genes (ttuA and ttuB; tRNA-two-thiouridine) that are essential for the synthesis of s2T in Thermus thermophilus. Mutation of either gene completely abolishes thio-modification of s2T, and these mutants exhibit a temperature-sensitive phenotype. These results suggest that bacterial growth at higher temperatures is achieved through the thermal stabilization of tRNA by a 2-thiolation modification. TtuA (TTC0106) is possibly an ATPase possessing a P-loop motif. TtuB (TTC0105) is a putative thio-carrier protein that exhibits significant sequence homology with ThiS of the thiamine synthesis pathway. Both TtuA and TtuB are required for in vitro s2T formation in the presence of cysteine and ATP. The addition of cysteine desulfurases such as IscS (TTC0087) or SufS (TTC1373) enhances the sulfur transfer reaction in vitro. The cellular components of thermophilic organisms have evolved to function efficiently in high temperature environments. tRNA is also thermostabilized through post-transcriptional modifications, such as 2-thioribothymidine (s2T) 2The abbreviations used are: s2T, 2-thioribothymidine; rT, ribothymidine; s4U, 4-thiouridine; LC, liquid chromatography; MS, mass spectroscopy; APM, [(N-acryloylamino)phenyl]mercuric chloride; Gm, 2′-O-methylguanosine; D, dihydrouridine; t6A, 6-threonylcarbamoyladenosine; m7G, 7-methylguanosine; Ψ, pseudouridine; m1A, 1-methyladenosine; i6A, 6-isopentenyladenosine; s2C, 2-thiocytidine; HPLC, high performance liquid chromatography; ORF, open reading frame. 2The abbreviations used are: s2T, 2-thioribothymidine; rT, ribothymidine; s4U, 4-thiouridine; LC, liquid chromatography; MS, mass spectroscopy; APM, [(N-acryloylamino)phenyl]mercuric chloride; Gm, 2′-O-methylguanosine; D, dihydrouridine; t6A, 6-threonylcarbamoyladenosine; m7G, 7-methylguanosine; Ψ, pseudouridine; m1A, 1-methyladenosine; i6A, 6-isopentenyladenosine; s2C, 2-thiocytidine; HPLC, high performance liquid chromatography; ORF, open reading frame. at position 54. s2T is a 2-thiolated derivative of 5-methyluridine (ribothymidine (rT)), which is located at position 54 in the T loop of almost all tRNAs in the eubacterium Thermus thermophilus (1Watanabe K. Shinma M. Oshima T. Nishimura S. Biochem. Biophys. Res. Commun. 1976; 72: 1137-1144Crossref PubMed Scopus (97) Google Scholar) and the archaea Pyrococcus furiosus (2Kowalak J.A. Dalluge J.J. McCloskey J.A. Stetter K.O. Biochemistry. 1994; 33: 7869-7876Crossref PubMed Scopus (177) Google Scholar). The 2-thiolation content of rT54 increases with cultivation temperature; in T. thermophilus cells grown at ≥80 °C, more than half of the tRNAs contain s2T54, whereas at 50 °C only a small proportion were thiolated at this position. Additionally, the melting temperature of tRNA increases concomitantly with s2T incorporation (1Watanabe K. Shinma M. Oshima T. Nishimura S. Biochem. Biophys. Res. Commun. 1976; 72: 1137-1144Crossref PubMed Scopus (97) Google Scholar). In an in vitro translation assay, 2-thiolated tRNA functions more efficiently at higher temperatures, whereas unthiolated tRNA functions optimally at lower temperatures (3Yokoyama S. Watanabe K. Miyazawa T. Adv. Biophys. 1987; 23: 115-147Crossref PubMed Scopus (61) Google Scholar). These results suggest that the proportion of tRNA containing s2T54 is a key factor in the adaptation of the thermophilic translation system to varying environmental temperatures. NMR analysis has elucidated the mechanism by which 2-thiolation of rT54 effects the thermal stabilization of tRNA (4Watanabe K. Yokoyama S. Hansske F. Kasai H. Miyazawa T. Biochem. Biophys. Res. Commun. 1979; 91: 671-677Crossref PubMed Scopus (40) Google Scholar). s2T adopts the C3′-endo-gg-anti conformation, as do all residues in A-form RNA, due to steric effects between the bulky 2-thiocarbonyl group and the 2′-hydroxyl group. The inherent rigidity of s2T54 stabilizes the interaction between the D and T loops of tRNA, resulting in thermostability of the tertiary structure (3Yokoyama S. Watanabe K. Miyazawa T. Adv. Biophys. 1987; 23: 115-147Crossref PubMed Scopus (61) Google Scholar). Although the function of s2T has been studied extensively, information on the biosynthesis of the 2-thiocarbonyl group of s2T is limited. In previous work, we undertook a preliminary characterization of the 2-thiolation reaction in a cell-free extract of T. thermophilus and found that a sulfur atom from cysteine or sulfate was incorporated into s2T (5Shigi N. Suzuki T. Terada T. Shirouzu M. Yokoyama S. Watanabe K. J. Biol. Chem. 2006; 281: 2104-2113Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The aims of this study were to isolate novel tRNA-binding proteins involved in the s2T modification of tRNA in T. thermophilus, to confirm their activity through mutation and in vitro analyses, and to determine whether or not the s2T modification of tRNA is required for growth at elevated temperatures. Strains—The strains used for this study were wild-type T. thermophilus HB27 and T. thermophilus NS0801, which lacks the gene for 4-thiouridine (s4U) biosynthesis (thiI) (5Shigi N. Suzuki T. Terada T. Shirouzu M. Yokoyama S. Watanabe K. J. Biol. Chem. 2006; 281: 2104-2113Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Both wild-type and mutant strains were cultivated in rich medium (6Takada T. Akanuma S. Kotsuka T. Tamakoshi M. Yamagishi A. Oshima T. Appl. Environ. Microbiol. 1993; 59: 2737-2739Crossref PubMed Google Scholar) at 70 °C without or with 30 μg/ml kanamycin, respectively, unless otherwise stated. Escherichia coli JM109 (7Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11410) Google Scholar) and TOP10 (Invitrogen) were used as hosts for the genetic manipulation of plasmids. Preparation of tRNA-bound Resin—The template for the in vitro transcription of tRNAIle was constructed using PCR to amplify the tRNA sequence under control of the T7 promoter (8Milligan J.F. Uhlenbeck O.C. Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (1006) Google Scholar). The oligonucleotides used for the construction of a plasmid bearing the tRNAIle gene are ile-temp-F (5′-ggccgaattc taatacgact cactataggg cgattagctc agctggttag agcgcacgcc tgataagcgt gag-3′) and ile-temp-R (5′-cgcgcaagct tggatggaag acctggtggg cgatggtgga cttgaaccac cgacctcacg cttatcagg-3′). The PCR products were cloned into the EcoRI and HindIII sites of pUC19. For in vitro transcription, the pair of primers ile-F (5′-taatacgact cactataggg cg-3′)/ile-R (5′-tggtgggcga tggtggactt g-3′) was employed to PCR-amplify the template plasmid pUC19-tRNAIle. Transcription of the tRNA gene was conducted at 37 °C for 3 h in a reaction mixture containing 40 mm HEPES-KOH (pH 7.8), 5 mm dithiothreitol, 1 mm spermidine, 8 mm MgCl2, 1 mm each NTP, 5 mm GMP, 50 μg/ml bovine serum albumin, 2 μg/ml template DNA, and T7 RNA polymerase (8Milligan J.F. Uhlenbeck O.C. Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (1006) Google Scholar). In order to prepare tRNA-bound resin, the 3′-ribose of the tRNA was oxidized to form a dialdehyde at room temperature, for 1 h in the dark, in a buffer containing 100 mm sodium acetate (pH 5.2), 10 mm sodium periodate, and 10 mm MgCl2. The oxidized tRNA was purified using a NAP 5 gel filtration column (Amersham Biosciences) and recovered by ethanol precipitation. Oxidized tRNA was biotinylated using Biotin (Long Arm) hydrazide (Vector), incubated in 100 mm sodium acetate (pH 5.2) and 10 mm MgCl2, at room temperature overnight in the dark. Unreacted biotin was removed by gel filtration using a NAP 5 column, followed by ethanol precipitation of the biotinylated tRNA. The precipitate was resuspended in H buffer (50 mm HEPES-KOH buffer (pH 7.6), 100 mm KCl, 10 mm MgCl2, 1 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride) and mixed with Streptavidin-Sepharose HP (Amersham Biosciences) for 1 h at room temperature. The excess tRNA (unbound fraction) was removed from the resin by washing three times with H buffer. About 35 μg of tRNA was immobilized on 10 μl of Streptavidin-Sepharose HP. Purification and Identification of tRNA-binding Proteins—A cell extract was prepared from an early log phase T. thermophilus HB27 culture. Cells were resuspended in H buffer with 10 mm KCl and then sonicated at 4 °C and centrifuged at 7,000 × g for 15 min. The supernatant was collected and centrifuged at 100,000 × g for 2 h, after which the supernatant was again collected and subjected to desalting using a Nap 25 gel filtration column (Amersham Biosciences). About 30 mg of gel-filtrated S100 was obtained from a 1-liter culture of T. thermophilus HB27 (∼1.4 g of cells). In order to remove nonspecific binding proteins, the desalted S100 fraction (2.5 mg) was mixed with Streptavidin-Sepharose HP and incubated for 10 min at room temperature, after which the resin was removed. The resultant fraction was then applied to tRNA-Sepharose (∼50 μg of bound tRNAIle) and incubated at room temperature for 20 min, followed by washing three times with H buffer. The resin-bound protein was mixed with an equal volume of sample buffer (125 mm Tris-HCl (pH 6.8), 2% SDS, 0.7 m 2-mercapthoethanol, and 0.02% bromphenol blue) and incubated at 95 °C for 5 min, prior to separation using SDS-PAGE. Proteins were visualized using Coomassie Brilliant Blue R-250, and bands of interest were excised and subjected to in-gel tryptic digestion as described previously (9Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7736) Google Scholar). Tryptic digests were analyzed using nanoflow-HPLC-electrospray ionization tandem mass spectrometry. This consisted of a DiNa splitless nanoflow HPLC system (KYA Technologies) and a 50 × 0.15-mm inner diameter packed ODS (3-μm particle size) capillary column (KYA Technologies), which are used for efficient separation of small amounts of peptide. Tryptic digest fragments were separated in 0.1% formic acid in water, using a linear gradient from 0 to 100% of 70% acetonitrile for 40 min at a flow rate of 500 nl/min. Ionization of the eluted peptides was performed using LCQ ion trap mass spectrometry (Thermo Electron), through electrospray ionization (metal nanosprayer S; GL Sciences). Proteins were identified using the MASCOT data base search engine (Matrix Science) and compared with the T. thermophilus HB27 genome data base (NC_005835 and NC_005838 on NCBI). Construction of ttuA::km and ttuB::km Strains—To construct the mutant strains ttuA::km (ttc0106::km) and ttuB::km (ttc0105::km) (NS2710 and NS2720, respectively), we PCR-amplified 0.5 kb from the 5′- and 3′-flanking regions of both target open reading frames (ORFs), using the T. thermophilus HB27 genome as a template. The htk cassette (kanamycin resistance gene) was amplified from pUC18-pJHK3 (10Hoseki J. Yano T. Koyama Y. Kuramitsu S. Kagamiyama H. J. Biochem. (Tokyo). 1999; 126: 951-956Crossref PubMed Scopus (142) Google Scholar). These fragments were designed with complementary sequences, enabling the 5′-flanking region, htk, and the 3′-flanking region to be ligated by PCR. The pairs of oligonucleotides used were as follows: ttc0106-5′-F (5′-ccgaggggtg gcttaggacc-3′)/ttc0106-5′-R (5′-gtaccatatc cgccgtcata tcagccctct ccgtctcctt gac-3′), ttc0106-3′-F (5′-gttaatcatg ttggttacgc atgtgggacg ccgtctacc-3)/ttc0106-3′-R (5′-gggtccacgt cgcagtagtc-3′), ttc0105-5′-F (5′-gacgtcctcg tggtccaggc-3′)/ttc0105-5′-R (5′-gtaccatatc cgccgtcata tcatctccgg gttcaggcca agc-3′), ttc0105-3′-F (5′-gttaatcatg ttggttacgt gcggggcgag gagctcctc-3′)/ttc0105-3′-R (5′-gtagcgcttg gagaggccgc-3′), and htk-F (5′-tgatatgacg gcggatatgg tac-3′)/htk-R (5′-cgtaaccaac atgattaac-3′). The sequences complementary to the htk gene cassette are underlined. The PCR fragments containing the htk cassette insertion were cloned into pCR-BluntII-TOPO (Invitrogen) and sequenced. Using the plasmids prepared as described above, T. thermophilus HB27 was transformed as previously described (11Koyama Y. Hoshino T. Tomizuka N. Furukawa K. J. Bacteriol. 1986; 166: 338-340Crossref PubMed Google Scholar), and transformants were selected on rich medium containing 300 μg/ml kanamycin. Homologous recombination was confirmed using PCR amplification, followed by restriction digestion analysis. Analysis of tRNA Modification—Cells were grown at 70 °C to late log phase, and total RNA was extracted using Isogen (Wako). The tRNA fraction was further purified using 10% PAGE containing 7 m urea. Total tRNA was digested by nuclease P1 (Yamasa) and bacterial alkaline phosphatase (Takara) in 20 mm HEPES-KOH (pH 7.5), at 37 °C for 10 h. The hydrolysate of RNA was analyzed using liquid chromatography/mass spectroscopy (LC/MS), as described previously (12Kaneko T. Suzuki T. Kapushoc S.T. Rubio M.A. Ghazvini J. Watanabe K. Simpson L. Suzuki T. EMBO J. 2003; 22: 657-667Crossref PubMed Scopus (93) Google Scholar). Temperature-dependent Growth Phenotypes of ttuA and ttuB Strains— Cells were cultivated in the rich medium (30 μg/ml kanamycin was added to the mutant strains) at 70 °C overnight. Diluted culture (A600 = 0.1) and serial dilutions (10-1, 10-2, 10-3, and 10-4) were spotted onto rich medium plates and incubated for 31 h at 60 °C, 17 h at 70 °C, 15 h at 75 °C, 24 h at 80 °C, and 32 h at 82 °C. Overexpression and Purification of TtuA, TtuB, IscS, and SufS Proteins— The gene encoding TtuA (TTC0106) of T. thermophilus HB27 was cloned into the NdeI and HindIII sites of pET22b(+) (Novagene), resulting in a C-terminal His6-tagged fusion protein. The oligonucleotides used were TTC0106-Nde-F (5′-agccagctCA TATGgtctgc aaggtctgcg ggc-3′) and TTC0106-Hin-R (5′-gagctAAGCT Taccggcacg gaggggcttc-3′). The sequences for restriction sites are shown in capital letters. The genes encoding TtuB (TTC0105), IscS (TTC0087), and SufS (TTC1373) of T. thermophilus HB27 were cloned into the NdeI and BamHI sites of pET15b (Novagene), resulting in N-terminal His6-tagged fusion proteins. The pairs of oligonucleotides used were TTC0105-Nde-F (5′-agccagctCA TATGagggtc gttctgcgcc-3′)/TTC0105-Bam-R (5′-ggcatGGATC Ctaccctccc gagatggc-3′), TTC0087-Nde-F (5′-agccagctCA TATGcggggc gtctacctgg actacgc-3′)/TTC0087-Bgl-R (5′-ggcatAGATC Tcaagcccgg gcccgggcca cggcc-3′), and TTC1373-Nde-F (5′-agccagctCA TATGgaccta agcgccctga gggaggac-3′)/TTC1373-Bam-R (5′-ggcatGGATC Cttatagcca ggcccggtac ttggcctc-3′), respectively. pETDuet-1 (Novagene) was used for the co-expression of the untagged TtuA and N-His6-TtuB. The NcoI-BamHI fragment of pET15b-TtuB (N-His6-TtuB) was inserted in the same restriction sites of pET-Duet-1, and ttuA was cloned into the NdeI and BglII sites. The pair of oligonucleotides used is TTC0106-Nde-F (see above) and TTC0106-Bgl-R (5′-gagctAGATC Ttaaccggca cggaggggct tc-3′). E. coli Rosetta (DE3) (Novagene) was transformed with the expression plasmids described above. Cultures were grown to A600 = 0.6, induced with isopropyl 1-thio-β-d-galactopyranoside (0.5 mm for TtuA, 1 mm for TtuB, 0.01 mm for co-expression of TtuA/B, and 0.1 mm for IscS and SufS), and expressed for a further 4 h at 37 °C. Cells expressing recombinant His-tagged TtuB, IscS, and SufS were harvested, suspended in K100 buffer (50 mm HEPES-KOH buffer (pH 7.6), 100 mm KCl, 10 mm MgCl2, 5% glycerol, and 7 mm 2-mercaptoethanol) with 0.2 mm phenylmethylsulfonyl fluoride, and gently sonicated. Lysates were separated using centrifugation at 6,000 × g, and the supernatants were collected and heat-treated at 70 °C for 15 min. A second centrifugation at 6,000 × g was performed, and the supernatant was applied to a Ni2+-nitrilotriacetic acid-agarose (Qiagen) column, washed with K100 buffer containing 1 m NH4Cl and 10 mm imidazole, and eluted with K100 buffer with 250 mm imidazole. Purified proteins were desalted using a NAP 25 gel filtration column and K100 buffer. Protein-containing fractions were concentrated with Centricon columns (YM3 for TtuB and YM30 for IscS and SufS; Millipore Corp.). The His-tagged fusion protein TtuA and the co-expressed TtuA/TtuB were purified as above, except for the addition of 500 mm KCl to all buffers and 200 mm imidazole to the buffer for gel filtration of TtuA in order to prevent precipitation. Centricon columns YM10 and YM3 were used for concentration of TtuA and TtuA/TtuB, respectively. Protein concentrations were determined using a Bio-Rad protein assay kit with a bovine serum albumin standard. Glycerol was added to the purified protein solutions to a final concentration of 30%, and each sample was stored at -30 °C. 2-Thiolation Assay with Recombinant Proteins—tRNAPhe from Saccharomyces cerevisiae (Sigma) was dephosphorylated using bacterial alkaline phosphatase (Takara), and T4 polynucleotide kinase (Toyobo) was used to label the 5′ terminus with [γ-32P]ATP (110 TBq mmol-1; Amersham Biosciences), followed by purification with denaturing 10% PAGE. The 2-thiolation assays were performed at 60 °C for 20 min in 20 μlof H buffer containing 5 mm ATP, 1 mm cysteine, 20 μm pyridoxal 5′-phosphate, 5′-labeled tRNAPhe (∼50,000 cpm/∼10 ng), 100 μg of desalted S100 (from the NS0801 strain that had been grown at 80 °C), and 30 pmol of the recombinant proteins. tRNA was recovered using Isogen (Wako), ethanol-precipitated, and separated using [(N-acryloylamino)phenyl]mercuric chloride (APM)-containing PAGE (100 μm APM) (13Igloi G.L. Biochemistry. 1988; 27: 3842-3849Crossref PubMed Scopus (146) Google Scholar, 14Shigi N. Suzuki T. Tamakoshi M. Oshima T. Watanabe K. J. Biol. Chem. 2002; 277: 39128-39135Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Gels were exposed to an imaging plate, followed by analysis using a BAS1000 bioimaging analyzer (Fuji Photo Systems). The 2-thiolation reaction with [35S]cysteine was performed as described above with the following modifications. Four μg of S. cerevisiae tRNAPhe and 20 μm [35S]cysteine (20 μCi) were added to 20 μl of reaction mixture. [35S]cysteine was purchased from American Radiolabeled Chemicals. RNAs were recovered using Isogen reagent and ethanol-precipitated. For alkaline treatment, the RNA sample was incubated at 37 °C for 1 h in 100 mm HEPES-KOH (pH 9.0). The sample was then subjected to 10% PAGE using gels containing 7 m urea, and the gels were then stained with EtBr. The gel was dried, exposed to an imaging plate, and analyzed using a BAS 1000 bioimaging analyzer (Fuji Photo Systems). For nucleoside analysis, labeled tRNAPhe was purified by PAGE, and 35S-labeled nucleosides were analyzed by an HPLC system as essentially described in the literature (5Shigi N. Suzuki T. Terada T. Shirouzu M. Yokoyama S. Watanabe K. J. Biol. Chem. 2006; 281: 2104-2113Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Fractions, which were collected every 37.8 s, were measured for absorbance at 280 nm and radioactivity. Purification of tRNA-binding Proteins from T. thermophilus Cell Extract—In order to identify the gene encoding the protein responsible for tRNA thiolation, we isolated the tRNA-binding proteins from a cell extract of T. thermophilus and identified the corresponding genes using proteomics. Transcript tRNAIle was biotinylated with ∼90% efficiency (data not shown) and immobilized on a streptavidin-Sepharose resin. The immobilized tRNAIle was used for the affinity purification of tRNA-binding proteins from a cell extract of T. thermophilus (Fig. 1). Samples were separated using a gradient SDS-PAGE. When compared, the sample that was affinity-purified with tRNA-Sepharose (lane 3) exhibited a different pattern from that of the cell extract prior to purification (lane 1), and little nonspecific binding to the resin alone was observed (lane 2). This suggests that tRNA-binding proteins were purified successfully. The major protein bands in lane 3 were excised and analyzed using mass spectrometry. A total of 108 proteins were identified from 2,204 ORFs of T. thermophilus HB27. Of these, ∼40% were predicted to interact with RNA (tRNA, rRNA, and mRNA); the remainder comprised DNA-binding proteins, biotin-binding proteins, metabolic pathway proteins, and proteins of unknown function. Among the RNA-binding proteins identified, we found 14 tRNA-binding proteins; tRNA nucleotidyltransferase (CCA-adding enzyme), elongation factor Tu, Ile-tRNA-synthetase, peptidyl-tRNA hydrolase, and 10 tRNA-modifying enzymes (Fig. 1B). We used the tRNAIle species for the purification experiment, since native tRNAIle has eight posttranscriptional modifications (15Horie N. Hara-Yokoyama M. Yokoyama S. Watanabe K. Kuchino Y. Nishimura S. Miyazawa T. Biochemistry. 1985; 24: 5711-5715Crossref PubMed Scopus (66) Google Scholar): 4-thiouridine at position 8 (s4U8); 2′-O-methylguanosine at position 18 (Gm18); dihydrouridine at position 20a (D20a); 6-threonylcarbamoyladenosine at position 37 (t6A37); 7-methylguanosine at position 46 (m7G46); s2T54; pseudouridine at position 55 (Ψ55); and 1-methyladenosine at position 58 (m1A58). With the exception of the enzymes for 2-thiolation of s2T and 6-threonylation of t6A, all of the other enzymes responsible for the modification of tRNAIle have been identified (i.e. ThiI for s4U8 (16Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (85) Google Scholar), TrmH for Gm18 (17Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (48) Google Scholar), Dus for D20a (18Bishop A.C. Xu J. Johnson R.C. Schimmel P. de Crecy-Lagard V. J. Biol. Chem. 2002; 277: 25090-25095Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), TrmB for m7G46 (19De Bie L.G. Roovers M. Oudjama Y. Wattiez R. Tricot C. Stalon V. Droogmans L. Bujnicki J.M. J. Bacteriol. 2003; 185: 3238-3243Crossref PubMed Scopus (61) Google Scholar), TrmFO for rT54 (20Urbonavičius J. Skouloubris S. Myllykallio H. Grosjean H. Nucleic Acids Res. 2005; 33: 3955-3964Crossref PubMed Scopus (90) Google Scholar), TruB for Ψ55 (21Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar), and TrmI for m1A58 (22Droogmans L. Roovers M. Bujnicki J.M. Tricot C. Hartsch T. Stalon V. Grosjean H. Nucleic Acids Res. 2003; 31: 2148-2156Crossref PubMed Scopus (79) Google Scholar)). Additionally, enzymes that are not involved in modification of tRNAIle were identified, such as TruD for Ψ13 (23Kaya Y. Ofengand J. RNA. 2003; 9: 711-721Crossref PubMed Scopus (92) Google Scholar), MiaA for 6-isopentenyladenosine at position 37 (i6A37) (24Dihanich M.E. Najarian D. Clark R. Gillman E.C. Martin N.C. Hopper A.K. Mol. Cell. Biol. 1987; 7: 177-184Crossref PubMed Scopus (96) Google Scholar), and TruA for Ψ38-40 (25Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google Scholar). Considering these results, enrichment of modification enzymes from the cell extract turned out to have been very efficiently achieved by our strategy. Identification of the Genes Essential for the 2-Thiouridylation Reaction— Among the proteins in the affinity-purified sample, we identified one belonging to the TtcA family (TTC0106) (Figs. 1A, 2A, and 3A). This protein family includes TtcA, an enzyme that is responsible for the biosynthesis of 2-thiocytidine (s2C) at position 32 of tRNA (26Jäger G. Leipuviene R. Pollard M.G. Qian Q. Björk G.R. J. Bacteriol. 2004; 186: 750-757Crossref PubMed Scopus (67) Google Scholar). The amino acid sequence of TTC0106 indicates that it is more closely related to the Group II TtcA family of proteins. These proteins possess a P-loop motif (Ser-Gly-Gly-Xaa-Asp-(Ser/Thr)) and four or five Cys-Xaa-Xaa-Cys (CXXC) motifs. In contrast, TtcA from Escherichia coli is included in Group I, which possess a P-loop motif and two CXXC motifs. The P-loop motif specifically binds ATP to form an adenylated intermediate; other tRNA-modifying enzymes, such as MnmA, ThiI, and TilS, also possess this motif (27Kambampati R. Lauhon C.T. Biochemistry. 2003; 42: 1109-1117Crossref PubMed Scopus (130) Google Scholar, 28Mueller E.G. Palenchar P.M. Protein Sci. 1999; 8: 2424-2427Crossref PubMed Scopus (47) Google Scholar, 29Soma A. Ikeuchi Y. Kanemasa S. Kobayashi K. Ogasawara N. Ote T. Kato J. Watanabe K. Sekine Y. Suzuki T. Mol. Cell. 2003; 12: 689-698Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). By the analysis of tRNA modification using liquid chromatography/mass spectroscopy (LC/MS), we were unable to detect s2C in the tRNA mixture of T. thermophilus under similar conditions to those required for detection of s2C from E. coli (data not shown). Thus, we believe that TTC0106 is not involved in s2C biosynthesis but is a candidate for s2T biosynthesis.FIGURE 3Multiple sequence alignments of TTC0106 (TtuA) (A) and TTC0105 (TtuB) (B) from various organisms. A, multiple sequence alignment of TtuA. The P-loop motif, CXXC motifs, and CXXH motif, are indicated by thick lines above the alignment. The sequence of TtcA from E. coli was included. B, multiple sequence alignment of TtuB. The C-terminal GG motif is indicated by a thick line above the alignment. ThiS sequences from E. coli and T. thermophilus (TTC0316) were included. The various organisms are indicated by the first two or three letters of the gene identification as follows. TTC, T. thermophilus HB27; AQ, Aquifex aeolicus; TM, T. maritima; PF, Pyrococcus furiosus; TK, T. kodakarensis; MJ, Methanococcus jannaschii; EC, E. coli; AF, Archaeoglobus fulgidus; PAB, Pyrococcus abyssi; PH, Pyrococcus horikoshii; MTH, Methanobacterium thermoautotrophicum.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TTC0105 was also affinity-purified using tRNA-Sepharose (Figs. 1A and 2B). It is a paralog of ThiS, a sulfur carrier protein essential for biosynthesis of the sulfur-containing cofactor, thiamine (30Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 31Lauhon C.T. Kambampati R. J. Biol. Chem. 2000; 275: 20096-20103Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Multiple alignment of TTC0105 and related sequences indicates that these proteins possess a conserved C-terminal Gly-Gly motif (Fig. 3B). The C terminus of ThiS is thiocarboxylated with a sulfur atom from cysteine, and then the sulfur atom is incorporated into the thiamine precursor (31Lauhon C.T. Kambampati R. J. Biol. Chem. 2000; 275: 20096-20103Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 32Settembre E. Begley T.P. Ealick S.E. Curr. Opin. Struct. Biol. 2003; 13: 739-747Crossref PubMed Scopus (94) Google Scholar). TTC0316 appears to be an ortholog of ThiS, and not only does the sequence closely resemble ThiS from E. coli (Fig. 3B), but in the T. thermophilus genome, it is located in a thiamine biosynthesis operon (ThiESGOC, unknown ORF, and ThiD; TTC0315-0321). The genes encoding TTC0105 and TTC0106 form a single operon (Fig. 4A) and are probably transcribed from a transcriptional start site immediately upstream of the TTC0105 coding region. To examine whether or not TTC0105 and TTC0106 are involved in s2T biosynthesis, we constructed insertion mutants of ttc0106 or ttc0105 and analyzed the modified nucleosides of unfractionated tRNA using LC/MS. In order to avoid polar effects in the ttc0105 insertion strain, ttc0106 was constructed so as to be transcribed together with a drug resistance gene. In both mutants, s2T (27.7 min) was completely absent, although its precursor rT (21.4 min) was detected (Fig. 4B); other nucleoside modifications remained unchanged. These results suggest that both TTC0105 and TTC0106 are involved in s2T biosynthesis. A small peak (27.5 min) from dinucleotide GmpG was observed immediately before the s2T peak and was derived from incomplete digestion of tRNA. This assignment was confirmed by the mass spectrum (m/z = 643) and by the disappearance of the peak in trmH, a Gm18-methylase mutant (data not shown). Thus, we have renamed the proteins TTC0106 and TTC0105 to tRNA two-thiouridine-synthesizing protein A and B (TtuA and TtuB), respectively, and the corresponding genes are now designated ttuA and ttuB. The precursor of s2T was identified as rT, suggesting that the 5-methyltransferase that modifies U at position 54 (TrmFO) (20Urbonavičius J. Skouloubris S. Myllykallio H. Grosjean H. Nucleic Acids Res. 2005; 33: 395