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Conserved Bases in the TΨC Loop of tRNA Are Determinants for Thermophile-specific 2-Thiouridylation at Position 54

2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês

10.1074/jbc.m207323200

1083-351X

Naoki Shigi, Tsutomu Suzuki, Masatada Tamakoshi, Tairo Oshima, Kimitsuna Watanabe,

Genomics and Phylogenetic Studies

2-Thioribothymidine (s2T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs. The 2-thiocarbonyl group of s2T54 is known to be responsible for the thermostability of tRNA. The s2T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus. Little is known about the biosynthesis of s2T, including the sulfur donor, modification enzyme, and the tRNA structural requirements. To characterize 2-thiolation at position 54 in tRNA, we constructed an in vivo expression system using tRNAAsp with an altered sequence and a host-vector for T. thermophilus. We were able to detectin vivo activity of s2T54 thiolase using phenyl mercuric gel electrophoresis followed by Northern hybridization. 2-Thiolation at position 54 was identified in the precursor form of the tRNA, indicating that 2-thiolation precedes tRNA processing. To ascertain the elements that determine 2-thiolation in tRNA, systematic site-directed mutagenesis was carried out using the tRNAAspgene. Conserved residues C56 and A58 were identified as major determinants of 2-thiolation, whereas tertiary interaction between the T and D loops and non-conserved nucleosides in the T loop were revealed not to be important for the reaction. 2-Thioribothymidine (s2T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs. The 2-thiocarbonyl group of s2T54 is known to be responsible for the thermostability of tRNA. The s2T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus. Little is known about the biosynthesis of s2T, including the sulfur donor, modification enzyme, and the tRNA structural requirements. To characterize 2-thiolation at position 54 in tRNA, we constructed an in vivo expression system using tRNAAsp with an altered sequence and a host-vector for T. thermophilus. We were able to detectin vivo activity of s2T54 thiolase using phenyl mercuric gel electrophoresis followed by Northern hybridization. 2-Thiolation at position 54 was identified in the precursor form of the tRNA, indicating that 2-thiolation precedes tRNA processing. To ascertain the elements that determine 2-thiolation in tRNA, systematic site-directed mutagenesis was carried out using the tRNAAspgene. Conserved residues C56 and A58 were identified as major determinants of 2-thiolation, whereas tertiary interaction between the T and D loops and non-conserved nucleosides in the T loop were revealed not to be important for the reaction. 2-thioribothymidine ribothymidine 1-methyladenosine 2′-O-methylguanosine expressed tRNA constructed from tRNAAsp of T. thermophilus HB8 [(N-acryloylamino)phenyl]mercuric chloride 4-thiouridine pseudouridine dihydrouridine tRNA(m5U54)-methyltransferase liquid chromatography/mass spectroscopy A characteristic structural feature of tRNA is post-transcriptional modification. The roles of modified nucleosides in tRNA function are important and wide-ranging. They are known to include codon recognition, reading-frame maintenance, stabilization of the tertiary structure, and serving as identity determinants for amino acid specificity (1Curran J.F. Grosjean H. Benne R. Modification and Editing of RNA. American Society for Microbiology, Washington, D. C.1998: 493-516Google Scholar). The melting temperatures of tRNAs from the extreme thermophileThermus thermophilus sp. are 3–10 °C higher than those of corresponding tRNA species from the mesophilic bacteriumEscherichia coli, a feature that cannot be explained solely by the higher G-C pair content in Thermus tRNAs (2Watanabe K. Oshima T. Iijima K. Yamaizumi Z. Nishimura S. J. Biochem. (Tokyo). 1980; 87: 1-13Crossref PubMed Scopus (72) Google Scholar). Analyses of modified nucleosides in tRNAs from T. thermophilus revealed a thermophile-specific sulfur-containing modified nucleoside that was identified as 2-thioribothymidine (s2T)1 (2Watanabe K. Oshima T. Iijima K. Yamaizumi Z. Nishimura S. J. Biochem. (Tokyo). 1980; 87: 1-13Crossref PubMed Scopus (72) Google Scholar, 3Watanabe K. Oshima T. Saneyoshi M. Nishimura S. FEBS Lett. 1974; 43: 59-63Crossref PubMed Scopus (64) Google Scholar), a 2-thiolated derivative of 5-methyluridine (ribothymidine (T)) located at position 54 in the T loop of almost all tRNAs (4Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (814) Google Scholar). Because s2T54 is also present in tRNA from hyperthermophilic Archaea such as Pyrococcus furiosus, which contains about 0.77 mol % of s2T when cultured at 100 °C (5Kowalak J.A. Dalluge J.J. McCloskey J.A. Stetter K.O. Biochemistry. 1994; 33: 7869-7876Crossref PubMed Scopus (178) Google Scholar), 2-thiolation of T54 is postulated to be a common modification responsible for the thermostabilization mechanism of tRNA in both thermophilic eubacteria and Archaea. The 2-thiolation of T54 increases along with elevation of the cultivation temperature without any changes in other modifications such as 1-methyladenosine at position 58 (m1A58) or 2′-O-methylguanosine at position 18 (Gm18) (6Watanabe K. Shinma M. Oshima T. Nishimura S. Biochem. Biophys. Res. Commun. 1976; 72: 1137-1144Crossref PubMed Scopus (99) Google Scholar); more than half of the tRNAs in T. thermophilusHB8 cells grown at more than 80 °C were found to contain s2T54 instead of T54, whereas at 50 °C only a small proportion had s2T. In addition, the tRNA melting temperature increased concomitantly with increases in the s2T content (6Watanabe K. Shinma M. Oshima T. Nishimura S. Biochem. Biophys. Res. Commun. 1976; 72: 1137-1144Crossref PubMed Scopus (99) Google Scholar). These findings indicate that 2-thiolation of T54 is responsible for the thermostability of T. thermophilus tRNA under diverse cultivation temperatures, thereby ensuring the thermal adaptation of protein synthesis. The temperatures of the inflection point in the specific CD signal (7Watanabe K. Oshima T. Nishimura S. Nucleic Acids Res. 1976; 3: 1703-1713Crossref PubMed Scopus (43) Google Scholar) and in the characteristic chemical shift in the NMR spectra of s2T in T. thermophilus tRNA (8Davanloo P. Sprinzl M. Watanabe K. Albani M. Kersten H. Nucleic Acids Res. 1979; 6: 1571-1581Crossref PubMed Scopus (85) Google Scholar) show a good match with the melting temperature monitored by UV absorbance, suggesting a close correlation between the local conformation of s2T54 and the structural stability of tRNA. The mechanism of tRNA structure stabilization conferred by s2T has been elucidated by proton NMR analysis (9Watanabe K. Yokoyama S. Hansske F. Kasai H. Miyazawa T. Biochem. Biophys. Res. Commun. 1979; 91: 671-677Crossref PubMed Scopus (40) Google Scholar); the ribose puckering of s2T preferentially takes the C3′-endo-gg-anti conformation as do all residues in A-form RNA because of the steric effect of the bulky 2-thiocarbonyl group toward the 2′-hydroxyl group. This inherent rigidity of s2T54 gives stability to the elbow region formed by D loop-T loop interaction, resulting in the thermostability of the overall tRNA tertiary structure (10Yokoyama S. Watanabe K. Miyazawa T. Adv. Biophys. 1987; 23: 115-147Crossref PubMed Scopus (61) Google Scholar). Although s2T54 is clearly a key modification for tRNA stability and function at elevated temperatures, information on its biosynthesis is very limited. Features that remain to be elucidated include the sulfur donor, modification enzymes or related genes, and the tRNA structural elements necessary for 2-thiolation. A genetic approach is, thus, indispensable for the characterization of this modification. Using the leuB gene as a selective marker, some of the present authors recently developed a host-vector system forT. thermophilus (11Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Crossref PubMed Google Scholar) that facilitates studies on the thermostability of proteins or RNAs from the bacterium. Here, we describe the detection of 2-thiolation activity in vivo in a reporter tRNA expressed using this T. thermophilushost-vector system. A systematic mutation analysis enabled us to characterize 2-thiolation in the maturation process of the tRNA and to identify the structural requirements for 2-thiolation at position 54. T. thermophilus TTY1 (a ΔleuBΔpyrE strain derived from T. thermophilus HB27) (11Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Crossref PubMed Google Scholar) was used as the host strain throughout.E. coli JM109, DH5α, and MC1061 were used as hosts for the genetic manipulation of plasmids. Rich and minimal media for T. thermophilus were used according to the literature (12Takada T. Akanuma S. Kotsuka T. Tamakoshi M. Yamagishi A. Oshima T. Appl. Environ. Microbiol. 1993; 59: 2737-2739Crossref PubMed Google Scholar). Uracil (20 μg/ml) was supplied to the minimal medium. T. thermophilus was cultured at 70 °C in all the experiments. A T. thermophilus HB8 tRNAAsp gene with an altered sequence (tRNAAsp*) was designed so that tRNAAsp* could be discriminated from the intrinsic tRNAAsp and other tRNAs in the cells by Northern hybridization (Fig. 1). The putative promoter/terminator sequences from the T. thermophilus HB8 tRNASer gene (GenBankTM accession number:X07394) were attached to the designed tRNAAsp* so it would be expressed in T. thermophilus cells. A synthetic DNA fragment comprising the tRNAAsp* operon (about 300 bp; Fig.1 A) was constructed from 6 DNA fragments in the following manner. The 5′-half of the operon was synthesized by the Klenow reaction with the primer set 5I-F and 5I-R, each of which has an overlapping 16-bp complementary sequence at its 3′ terminus. The 3′-half was synthesized in the same way with the primers 3I-F and 3I-R. These two half-fragments were ligated using the primers 5F and 3R, respectively corresponding to the 5′- and 3′-termini of the operon. The ligated DNA fragment was then introduced into the pCR-XL-TOPO vector (Invitrogen). To confirm the correctness of the construction, the tRNA operon in the resultant plasmid (pCR-XL-tRNAAsp*) was sequenced by an ABI PRISM 3100 DNA sequencer (Applied Biosystems). A tRNA expression plasmid for T. thermophilus (pEx-Asp*) was constructed by excising the tRNAAsp* gene from pCR-XL-tRNAAsp* by double digestion with EcoRI and EcoRV and introducing it into the same sites of theE. coli-T. thermophilus shuttle vector pT8leuB(11Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Crossref PubMed Google Scholar). T. thermophilus TTY1 (11Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Crossref PubMed Google Scholar) was transformed by the tRNA expression vectors as described in the literature (13Koyama Y. Hoshino T. Tomizuka N. Furukawa K. J. Bacteriol. 1986; 166: 338-340Crossref PubMed Google Scholar). The transformants were selected on minimal medium plus uracil plates at 70 °C. The primers used to construct the tRNAAsp* gene were: 5I-F (82-mer), 5′-CTCCCCGGGT AAAGCCGCCC CGCCAAGATC ATGGAGCCCA AGGCGTCAAA GTCCAAGTTT TCGTGGGCCG CCACGACCCG CA-3′; 5I-R (86-mer), 5′-CACCAACACC GCAAGTGCCA AAAAGGAGTG TACCAGCGCC GTCCGCCTCC GTCAAGGTAG ACTGGGGCCG TGCGGGTCGT GGCGGC-3′; 3I-F (83-mer), 5′-TGGCACTTGC GGTGTTGGTG TAGTTGGTTA ACACACGGTC CTGTCACGAC CGAGATCGCC CGTTCGAGTC GGGTACACCG CGC-3′; 3I-R (86-mer), 5′-CCCAAAGGGC CCCGGAGGCG GCGCGGGCCC ACGGGAAAAG GGAGCCCCGG CCTTCGCCGG GGCCTTTTGG CGCGGTGTAC CCGACT-3′; 5F (36-mer), 5′-ATCATGATAT CCTCCCCGGG TAAAGCCGCC CCGCCA-3′; 3R (35-mer), 5′-GCATTGAATT CCCCAAAGGG CCCCGGAGGC GGCGC-3′. Plasmids harboring 24 tRNA variants were prepared using a QuikChangeTM site-directed mutagenesis kit (Stratagene). Target mutations were introduced into the plasmids using 30-mer forward primers and the complementary reverse primers, both of which possessed the target mutation in the middle section. pCR-XL-tRNAAsp* and pCR-XL-tRNAAsp*(U8A) were exploited as template plasmids for mutagenesis. After the sequences of these tRNA variants were confirmed by a DNA sequencer (as above), the tRNA operons were inserted into pT8leuBto construct the respective tRNA expression vectors. Total RNAs were extracted from cultured cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method as described in the literature (14Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63148) Google Scholar) and subjected to electrophoretic analysis using a 10% polyacrylamide gel containing 7m urea. The RNA bands separated in the gel were transferred to a nylon membrane (Hybond N+, Amersham Biosciences). Northern blotting was performed using the following 5′32P-labeled oligonucleotide probes specific for tRNAAsp* and its precursors: AS-1 (34-mer), 5′-CGCGGTGTAC CCGACTCGAA CGGGCGATCT CGGT-3′; AS-2 (17-mer), 5′-CGCGGTGTAC CCGACTC-3′; AS-5′ (15-mer), 5′-TACACCGCAA GTGCC-3′; AS-3′ (25-mer), 5′-TCGCCGGGGC CTTTTGGCGC GGTGT-3′. The positions of AS-1, AS-5′, and AS-3′ are shown in Fig. 1 B. Membranes on which 32P-labeled oligonucleotide probes hybridized with the corresponding tRNAs and their precursors were exposed to an imaging plate followed by analysis using a BAS1000 bioimaging analyzer (Fuji Photo Systems). About 1000 A 260 units of total RNA were obtained by the acid guanidinium thiocyanate-phenol-chloroform extraction method (14Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63148) Google Scholar) from a 3L late log-phase culture of the TTY1 transformant expressing tRNAAsp*. Total RNA was fractionated on a DEAE-Sepharose fast flow column (1 × 40 cm) with a linear gradient of NaCl and MgCl2 consisting of 500 ml of elution buffer A (20 mm Tris-HCl (pH 7.5), 200 mm NaCl, and 8 mm MgCl2) and elution buffer B (20 mm Tris-HCl (pH 7.5), 450 mm NaCl, and 16 mm MgCl2) with gravitational flow. Fractions containing tRNAAsp* were determined by dot-hybridization using the DNA probe AS-1, combined, precipitated with ethanol, and dissolved in a binding buffer (1.2 m NaCl, 30 mm Tris-HCl (pH 7.6) and 15 mm EDTA). To procure tRNAAsp* on its own, the solid-phase DNA probe method (15Wakita K. Watanabe Y. Yokogawa T. Kumazawa Y. Nakamura S. Ueda T. Watanabe K. Nishikawa K. Nucleic Acids Res. 1994; 22: 347-353Crossref PubMed Scopus (75) Google Scholar) was employed using a 3′-biotinylated oligonucleotide with a sequence identical to that of the DNA probe AS-1. The tRNAAsp* obtained was further purified by denaturing polyacrylamide gel electrophoresis. Purified tRNAAsp* was sequenced by the method of Donis-Keller (16Donis-Keller H. Nucleic Acids Res. 1980; 8: 3133-3142Crossref PubMed Scopus (258) Google Scholar). Partial enzymatic digestion was carried out with the following base-specific RNases: RNase T1 (Amersham Biosciences), RNase U2 (Seikagaku Kogyo), RNase PhyM (Amersham Biosciences), and RNase CL3(Roche Molecular Biochemicals). Digested fragments were electrophoresed separately in lanes on a 15% denaturing polyacrylamide gel along with the undigested control and alkaline-hydrolyzed tRNAs. Modified nucleotides were identified by the post-labeling method (17Kuchino Y. Hanyu N. Nishimura S. Methods Enzymol. 1987; 155: 379-396Crossref PubMed Scopus (74) Google Scholar). An LCQ ion trap mass spectrometer (ThermoFinnigan) equipped with an electrospray ionization source and a MAGIC 2002 liquid chromatography system (Michrom BioResources) was used to analyze RNA fragments digested with RNase T1. Purified tRNAAsp* (0.4 μg) was digested with RNase T1 (2.5 units) in 25 mm ammonium acetate (pH 5.3) at 37 °C for 1 h, and the digest was subjected to mass spectrometric analysis. Oligonucleotides produced by RNase T1 digestion were detected by LC/MS in the negative ion mode as described by Qiu and McCloskey (18Qiu F. McCloskey J.A. Nucleic Acids Res. 1999; 27: e20Crossref PubMed Google Scholar) with the following slight modifications. An ODS reversed-phase column (Inertsil ODS3, 1.0 × 200 mm; GL Sciences) was used. The solvent system, consisting of 0.4 m 1,1,1,3,3,3-hexafluoro-2-propanol (pH 7.0, adjusted with triethylamine) in H2O (A) and 50% methanol (B) was used as follows: 15–95% B in 0–15 min, 95% B for 15–25 min, 95–15% B in 25–26 min. Negative ions were scanned over an m/z range from 620 to 2000. The 2-thiolation at position 54 in tRNAAsp* and its precursor were detected by retardation in an electrophoretic system consisting of a 10% polyacrylamide gel (10 × 12 × 0.1 cm3) containing 7 m urea polymerized in the presence of 25 or 100 μm[(N-acryloylamino)phenyl]mercuric chloride (APM), which was synthesized as described by Igloi (19Igloi G.L. Biochemistry. 1988; 27: 3842-3849Crossref PubMed Scopus (148) Google Scholar). Total RNA (1.6–3.2 μg) was resolved on the APM gel. After electrophoresis, the gel was soaked in 0.2 m β-mercaptoethanol for 1 h to reduce and/or break the sulfur linkages formed between the thiolated-RNA and the APM in the gel. The RNA was transferred onto a nylon membrane (Hybond-N+, Amersham Biosciences) by blotting using TBE Buffer, which was hybridized with the 5′-32P-labeled DNA probes for tRNAAsp* or its precursor. The retarded band resulting from the presence of thiocarbonylated nucleotides in RNA was visualized by a Fuji BAS1000 bioimaging analyzer. The 5′ end of the precursor tRNAAsp* was determined by the primer extension technique. Total RNA from TTY1/pT8leuBor TTY1/pEx-Asp* (U8A/G19C) was heat-denatured and annealed with the 5′-32P-labeled primer RT-1 (15-mer), 5′-CAACTACACC TACAC-3′. Reverse transcription was performed with Moloney murine leukemia virus reverse transcriptase (Toyobo) according to the manufacturer's instructions. To determine the initiation position of the transcription, the primer extension product was electrophoresed together with the dideoxy sequencing products of the template plasmid pCR-XL-tRNAAsp*(U8A) using the same primer (RT-1) on a 15% denaturing polyacrylamide sequencing gel. To detect activity of the s2T (54)-thiolase in T. thermophilus cells and to investigate its recognition elements in tRNA in vivo, we first expressed an artificial tRNA gene encoded on a plasmid. We selected T. thermophilus HB8 tRNAAsp as our model tRNA species because the presence of s2T at position 54 has been verified in this tRNA (20Keith G. Yusupov M. Briand C. Moras D. Kern D. Brion C. Nucleic Acids Res. 1993; 21: 4399Crossref PubMed Scopus (17) Google Scholar). We introduced mutations at 11 base pairs in the acceptor, anticodon, and T stems of tRNAAsp (Fig.1 B) so that the resultant tRNA (named tRNAAsp*) could be discriminated from the native tRNAAsp by Northern hybridization using the DNA probes for tRNAAsp*. The tRNAAsp* gene was integrated into the tRNASer operon from T. thermophilus strain HB8 by replacing the tRNASer gene, as depicted in Fig. 1,A and B). The reason for employing this strategy is that the tRNASer operon is the only T. thermophilus tRNA gene reported so far that includes both 5′- and 3′-adjacent apparent promoter and terminator sequences. The tRNAAsp* operon was synthesized by PCR using six DNA oligos and ligated into the EcoRI and EcoRV sites of anE. coli-T. thermophilus shuttle vector, pT8leuB(11Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Crossref PubMed Google Scholar), resulting in an expression vector for tRNAAsp* (pEx-Asp*). Total RNA from cells of E. coli JM109 harboring pEx-Asp* was separated by 10% denaturing PAGE followed by Northern hybridization with the probe AS-2. Two distinct RNA bands associated with tRNAAsp* could be observed (Fig.2 A). The longer band (asterisk), which appeared in close proximity to 5 S rRNA, was considered to be a precursor of tRNAAsp*. The shorter band, located in the E. coli class I tRNA cluster, was presumed to be the mature form of tRNAAsp*. The result demonstrated that the tRNA operon from T. thermophilus could function even in E. coli cells, which can be explained by the fact that the promoter sequence of this gene (TTGACG (−35)/TACACT (−10) (Fig. 1 A) is similar to the E. coli tRNA promoter consensus sequence (TTGACA (−35)/TATAAT (−10) (21Inokuchi H. Yamao F. Söll D. RajBhandary U. tRNA: Structure, Biosynthesis, and Function. American Society for Microbiology, Washington, D. C.1995: 17-30Google Scholar). However, the heterologous expression of Thermus tRNA in E. coli cells resulted in strong accumulation of the tRNA precursor (∼70% of the total expression), indicating that the slight disparity with the consensus sequence and the high copy number of the shuttle vector derived from pUC118 in E. coli cells (22Yanisch-Perron C. Vieira J. Messing J. Gene. 1985; 33: 103-119Crossref PubMed Scopus (11458) Google Scholar, 23Vieira J. Messing J. Methods Enzymol. 1987; 153: 3-11Crossref PubMed Scopus (2007) Google Scholar) may have led to the tRNA precursor being produced too abundantly to be processed sufficiently by tRNA maturases such as RNase P (24Altman S. Kirsebom L. Talbot S. Söll D. RajBhandary U. tRNA: Structure, Biosynthesis, and Function. American Society for Microbiology, Washington, D. C.1995: 67-78Google Scholar) or 3′ ribonucleases (25Deutscher M.P. Li Z. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 67-105Crossref PubMed Scopus (118) Google Scholar). When the expression of tRNAAsp* in T. thermophilus was examined by Northern hybridization with the probe AS-1 (Fig. 2 B), tRNAAsp* originating from the vector was detected mainly in the mature form, although as much as 30% of the total product was observed as the putative precursor (asterisk). Judging from the intensity of the Northern blotting, the expression of total RNA in T. thermophiluscells was lower than that in E. coli cells. This can be accounted for by the likely low copy number of pEx-Asp* in T. thermophilus, because this expression vector has a replication origin derived from pTT8, whose copy number is about eight (26Hishinuma F. Tanaka T. Sakaguchi K. J. Gen. Microbiol. 1978; 104: 193-199Crossref PubMed Scopus (41) Google Scholar). Our next task was to determine whether tRNAAsp* expressed in T. thermophilus cells was processed normally and modified in the same manner as the native tRNA and, in particular, whether or not it contained s2T. To isolate tRNAAsp*, total RNA from T. thermophiluscells harboring pEx-Asp* was fractionated by anion exchange column chromatography so as to enrich the fractions that included tRNAAsp*. In this step, we were able to successfully concentrate the tRNAAsp* fractions and exclude those containing the precursor. tRNAAsp* was then purified to homogeneity by solid-phase DNA probing (Fig.3 A). End-labeling with32P and sequencing by Donis-Keller's enzymatic digestion method (Fig. 3 B) showed that the purified tRNAAsp* had the expected sequence, including both the 5′ and 3′ ends. Abnormalities in the bands suggested the presence of some modified residues (the expected modifications are parenthesized in Fig.3 B). The primary sequence of tRNAAsp* was further determined by post-labeling and LC/MS analysis, which enabled us to identify 7 post-transcriptional modifications at 8 positions: 4-thiouridine (s 4 U) at position 8, pseudouridine (Ψ) at positions 13 and 55, 2′-O-methylguanosine (Gm) at position 18, dihydrouridine (D) at positions 20 and 20a, 1-methyladenosine (m 1 A) at position 58, ribothymidine (T) at position 54, and 2-thioribothymidine (s 2 T) at position 54 (Fig.3 C). The post-labeling method clearly identified both T and s2T at position 54 (data not shown), which is consistent with a previous report that s2T is derived from a partial modification of T induced by the cultivation temperature (6Watanabe K. Shinma M. Oshima T. Nishimura S. Biochem. Biophys. Res. Commun. 1976; 72: 1137-1144Crossref PubMed Scopus (99) Google Scholar). The presence of s2T at position 54 was further examined by LC/MS with RNase T1-digested fragments of tRNAAsp* (Fig. 4). Although the fragment s2TΨCGp was clearly detected at retention time 20.1 min as singly and doubly charged ions, the fragment TΨCGp was hardly detected even though the LC/MS was highly sensitive, suggesting that tRNAAsp* preferentially possesses s2T at position 54.Figure 4Mass spectrometric analysis of oligonucleotides derived from tRNAAsp*. A, mass chromatograms of oligonucleotides obtained by RNase T1digestion of tRNAAsp*. The upper panel shows base peaks in the mass chromatogram of m/z = 620–2000, within which each oligonucleotide was detected. Peak numbers represent RNA fragments derived from tRNAAsp*:1, m1AGp; 2, CGp; 3, UGp;4, AGp; 5, ΨAGp; 6, UCGp;7, UUGmGp; 8, ACCGp; 9, AUCGp;10, UCCUGp; 11, UCACGp; 12, UACACCGp;13, Us4UGp; 14, DDAACACACGp. Themiddle and lower panels show mass chromatograms of m/z = 1309 andm/z = 654 for [M-H]− and [M-2H]2− ions of s2TΨCGp, respectively.B, mass spectrum in the range 19.64–20.50 min. [M-H]− and [M-2H]2− ions of s2TΨCGp were detected. Single and double asterisks, respectively, indicate the [M-H]− ion and [M-2H]2− ion peaks derived from UUGmGp, which gave the same retention time.View Large Image Figure ViewerDownload (PPT) Our experimental results demonstrated that tRNAAsp* was expressed in T. thermophilus cells in the mature form and that it carried 8 modifications including s2T at position 54. We thus successfully detected s2T synthesis activityin vivo. Because the procedures we employed to detect s2T54 are unsuitable for routine assays of multiple samples, we searched for a simpler method that does not require purification of the expressed tRNA. Igloi (19Igloi G.L. Biochemistry. 1988; 27: 3842-3849Crossref PubMed Scopus (148) Google Scholar) reports an affinity electrophoresis system in which a polyacrylamide gel is co-polymerized with APM. In this system, which was reported to be successful in detecting tRNA with the s4U or 5-methylaminomethyl-2-thiouridine modification (19Igloi G.L. Biochemistry. 1988; 27: 3842-3849Crossref PubMed Scopus (148) Google Scholar), the electrophoretic mobility of thiolated tRNAs is retarded compared with that of non-thiolated tRNAs due to the specific interaction between the thiocarbonyl group and the mercuric compound. By combining this technique with Northern hybridization using a probe specific for the relevant tRNA, the thiolated nucleosides in a particular tRNA species can be detected (27Emilsson V. Naslund A.K. Kurland C.G. Nucleic Acids Res. 1992; 20: 4499-4505Crossref PubMed Scopus (38) Google Scholar). We therefore used this approach to identify the 2-thiouridylation at position 54 in tRNAAsp* expressed inT. thermophilus cells. When APM gel electrophoresis was performed, specific retardation of tRNAAsp* was observed (data not shown). However, because tRNAAsp* has 2 thiouridines, s4U8 and s2T54, this retardation would have been mediated by both of them. To differentiate gel retardation due to 2-thiolation at position 54 from that caused by s4U8, we first eliminated s4U8 from the expressed tRNAAsp* by introducing a U to A point mutation at position 8 in the expression vector pEx-Asp*. As shown in Fig. 5 A(lanes 1 and 4), the mutant tRNAAsp*(U8A) without the s4U8 modification was expressed in T. thermophilus cells in its mature form together with a few possible precursors from the resultant plasmid (pEx-Asp*(U8A)). Total RNA from this mutant strain was subjected to APM gel electrophoresis using two APM concentrations, 25 and 100 μm (+ and ++, respectively, in Fig. 5). In the gels stained by ethidium bromide (lanes 2 and 3), most of the intrinsic tRNAs exhibited strong retardation due to s4U8. Furthermore, when compared with the 5 S rRNA band, which contains no thiolated nucleotides, the tRNA cluster was retarded in accordance with the concentration of APM. Next, the band corresponding to the expressed tRNAAsp*(U8A) was detected by Northern hybridization using the probe AS-1 (Fig. 5 A,lanes 4–6). Slight but significant retardation concomitant with the increment in the APM concentration could be observed. This was thought to be almost certainly caused by the presence of the 2-thio group at position 54 in tRNAAsp*(U8A) given that band retardation due to 2-thiolation at position 54 is likely to be much less than the strong retardation resulting from 4-thiolation of U8. This interpretation is supported by the reported successful detection of the other 2-thiouridine derivative, 5-methylaminomethyl-2-thiouridine, by the APM method (19Igloi G.L. Biochemistry. 1988; 27: 3842-3849Crossref PubMed Scopus (148) Google Scholar). To confirm that the observed band retardation did actually derive from 2-thiolation at position 54, we constructed another mutant vector, tRNAAsp*(U8A/U54A), in which a U to A point mutation was introduced at position 54 in addition to that at position 8. As shown in Fig. 5 B (lanes 7 and 10), tRNAAsp*(U8A/U54A) was expressed in its mature form together with a large accumulation in the precursor form, indicating that the U54A mutation had an inhibitory effect on tRNA maturation. In the presence of APM, the bands corresponding to the mature form of tRNAAsp* (U8A/U54A) (lanes 11 and 12) appeared at the same position as the bands for tRNAAsp*(U8A) and tRNAAsp*(U8A/U54A) on the gels without APM (lanes 4 and 10, respectively). Moreover, no difference in mobility was observed even when the concentration of APM was increased (lanes 11 and12). The above results clearly demonstrated that the retardation of tRNAAsp*(U8A) in APM gel electrophoresis actually resulted from the presence of the 2-thio group at position 54 in the tRNA and confirmed the feasibility of using this simple assay system to investigate the sequence requirements of tRNA for in vivo2-thiolation