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. 2010 Nov 18;39(6):2304–2318. doi: 10.1093/nar/gkq1180

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus

Kazuo Ishida 1, Takashi Kunibayashi 1, Chie Tomikawa 1, Anna Ochi 1, Tamotsu Kanai 2, Akira Hirata 1, Chikako Iwashita 1, Hiroyuki Hori 1,3,4,*
PMCID: PMC3064792  PMID: 21097467

Abstract

Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50°C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50°C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m5s2U54 and m1A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. 35S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C.

INTRODUCTION

Pseudouridine (Ψ) is the most abundant modified nucleoside in RNA and is frequently found in tRNA, rRNA, snRNA, snoRNA and tmRNA (1–6). Ψ is formed post-transcriptionally via C5-ribosyl isomerization of uridine by a class of enzymes known as Ψ synthases. Most Ψ synthases recognize one or a few target uridines and catalyze the isomerization.

Transfer RNA (Ψ55) synthase [EC.5.4.99.12 (7); TrPus(Y55) (1)] catalyzes Ψ55 formation in the T-loop of tRNA. The Ψ55 modification is commonly found in eubacterial, archaeal, eukaryotic and organelle tRNA (1–3). The Ψ55 forms a tertiary base pair with the conserved G18 in D-loop and stabilizes the L-shaped tRNA structure (8,9). Eubacterial tRNA (Ψ55) synthase is one of the best studied tRNA modification enzymes. The Ψ formation activity towards a tRNA precursor was first detected in Escherichia coli cell extract (10) and subsequently tRNA (Ψ55) synthase activity was separated from tRNA (Ψ38, 39, 40) synthase activity (11). The tRNA (Ψ38, 39, 40) synthase is now called tRNA Ψ synthase I (TruA) (7,12–14). To date, three tRNA Ψ synthases (TruC, RluA and TruD) have been additionally found in E. coli (15,16). E. coli tRNA (Ψ55) synthase was purified to near homogeneity as judged by SDS–polyacrylamide gel analysis, and found to be encoded by the truB gene (17). Therefore, hereafter we describe eubacterial tRNA (Ψ55) synthase as TruB. It should be mentioned that the Ψ55 in archaeal tRNA is formed by Cbf5 (18–20) and Pus10 (18,21), and that Ψ55 in eukaryotic cytoplasmic and mitochondrial tRNA is formed by Pus4 (22). Although the truB genes are found in almost all eubacterial genomes (23,24), the gene has been experimentally identified in E. coli (17), Bacillus subtilis (17), Shigella flexneri (25), Pseudomonas aeruginosa (25,26) and Thermotoga maritima (27,28). TruB recognizes the T-arm structure and can modify the target uridine in a 17-mer T-arm fragment (29). X-ray crystal structures of TruB and TruB-RNA complex have been studied and have revealed that TruB undergoes significant conformational changes upon binding to RNA substrate (27,30–32). Based on amino acid sequence alignment and crystal structures, the mechanism of uridine isomerization has also been studied (23,24,28,30,31,33–36) and the catalytic residue of TruB been identified as a conserved aspartic acid (34). Thus, in vitro protein and RNA chemistry studies have made significantly progress in the past 15 years since the identification of the truB gene.

In vivo functional study of the Ψ55 modification in tRNA is not straightforward because a truB deletion mutant generally grows normally. For example, deletion of E. coli truB does not affect exponential growth (37) and the lack of the Ψ55 modification does not cause frameshift error on translational ribosomes in E. coli cells (38). However, a large effort has been made toward elucidating the importance of TruB protein and the Ψ55 modification. Thus, the translational activity of nine-repeated CGA codons in the E. coli truB deletion mutant is inferior to that in the wild-type strain (25). The E. coli truB deletion mutant exhibits a defect in survival of rapid transfer from 37°C to 50°C (39). Deletion of truB in S. flexneri reduces the expression of some virulence-associated genes (25). Disruption of truB (classical name, orp gene) by Tn5 in P. aeruginosa results in impaired growth on BHI plates at 43°C and a reduced amounts of virulence factor phospholipase C compared to the wild-type strain (25,26). Deletion of truB alone in E. coli does not affect the suppressor tRNA activity of tyrT (supF), however increased suppression ability in truB–trmH and truB–trmA double deletion mutants is observed (25). Furthermore, these double deletion mutants show reduced growth (25). The genes, trmH and trmA encode tRNA (Gm18) methyltransferase (24,40–46) and S-adenosyl-l-methionine (AdoMet)-dependent tRNA (m5U54) methyltransferase (47–51), respectively. These facts suggest that Ψ55 modification probably works with other modifications (Gm18 and m5U54) at the elbow region in the L-shaped tRNA structure and that Ψ55 modification and/or TruB protein itself is required for stress resistance. In these functional studies, a very interesting hypothesis has been proposed namely that TruB protein may act as an RNA chaperone to assist in correct folding of tRNA (37).

In this article, we found that a truB disruptant strain of Thermus thermophilus, an extreme-thermophilic eubacterium, showed growth retardation at low temperatures (below 60°C). We demonstrate that the lack of the Ψ55 modification causes abnormal enhancement of the amount of other modified nucleotides. We describe in this article that the Ψ55 modification is required for low temperature adaptation for the organism. Furthermore, the RNA chaperone hypothesis of the TruB protein is discussed.

MATERAILS AND METHODS

Materials

[α-32P]-Uridine 5′-triphosphate (222 TBq/mmol; 0.37 MBq/µl) was purchased from MP Biomedicals. [Methyl-14C]-AdoMet (1.95 GBq/mmol) and [methyl-3H]-AdoMet (2.47 TBq/mmol) were purchased from Perkin Elmer. l-[35S]-Methionine (37 TBq/mmol) was purchased from Muromachi Yakuhin. DE52 is a product of Whatman. CM-Toyopearl 650 M was bought from Tosoh. DNA oligomers were obtained from Invitrogen and T7 RNA polymerase was from Toyobo. Other chemical reagents were of analytical grade.

Strain and media

Culture source of T. thermophilus HB8 was a kind gift from Dr Tairo Oshima (Tokyo University of Pharmacy and Life Science). The cells were grown in rich medium [0.8% polypeptone, 0.4% yeast extract and 0.2% NaCl, pH 7.5 (adjusted with NaOH)]. The medium was supplemented with 0.35 mM CaCl2 and 0.17 mM MgCl2 after autoclaving. To make plates, gellan gum (Wako Pure Chemicals) was added to the medium (final concentration, 1.5%).

Selection of a target gene for gene disruption and preparation of the recombinant TruB protein

The TTHA0217 gene in T. thermophilus HB8 genome has been annotated as truB by the T. thermophilus DNA sequencing project (52). A DNA fragment of the TTHA0217 gene was amplified by PCR using following primers: Tth TruBF, 5′-CCC CCA TAT GGC CCT CTA CGC GGT GGA CAA G-3′; Tth TruBR, 5′-CCC CGG ATC CTT ACG CCT CCT TCG CAA ACA CGC C-3′. Underlining shows restriction enzyme sites (Nde I and Bam HI). The amplified DNA was digested with Nde I and Bam HI, and ligated into the multi-cloning linker of pET30a E. coli expression vector. The expression of TTHA0217 protein in E. coli BL21 (DE3) Rosetta 2 strain (Novagen) was performed according to the manufacturer’s manual. The protein was purified by DE52 column chromatography, heat-treatment and CM-Toyopearl 650 M column chromatography. Briefly, the cells (5g) were suspended in 25 ml of buffer A [50 mM Tris–HCl (pH 7.6), 5 mM MgCl2, 6 mM 2-mercaptoethanol and 50 mM KCl] and disrupted with an ultrasonic disruptor model UD-200 (Tomy, Japan). The cell debris was removed by centrifugation at 8000g for 20 min. The supernatant fraction was applied onto a DE52 column (column volume, 10 ml). The flow-through fractions were collected and then heated at 70°C for 30 min. The denatured proteins were removed by centrifugation at 8000g for 20 min. The supernatant fraction was dialyzed against buffer B [50 mM Hepes-KOH (pH 6.8) and 6 mM 2-mercaptoethanol]. The dialyzed sample was loaded onto a CM-Toyopearl 650M column (column volume, 10 ml) and TruB protein eluted with buffer B containing 100–150 mM KCl. The eluted sample was dialyzed against buffer A and concentrated with Centriprep YM-10 centrifugal filter devices (Millipore). Glycerol was added to the purified protein to a final concentration of 50% v/v and stored at −30°C. Quantification of protein was performed with a Bio-Rad protein assay kit using bovine serum albumin as standard.

Measurement of Ψ formation activity

We selected T. thermophilus tRNAThr (GGT) as the substrate of TruB because this tRNA is expected to possess only one Ψ modification at position 55 (2). Template DNA for transcription was prepared with the following primers: Tth tRNAThr F, 5′-TAA TAC GAC TCA CTA TAG CTC GCG TAG CTC AGC AGG TAG AGC ACA CCC TTG TAA GGG TG-3′; Tth tRNAThr R, 5′-TGG AGC TCG CGG CCG GGC TCG AAC CGG CCC TCA CGC TTA CCA AGG GTG TG-3′. Run-off transcription by T7 RNA polymerase was performed according to our previous reports (42,53). In the case of internal labeling, 2 µl of α-32P-UTP was added into the reaction mixture. The transcript was purified by 10% PAGE (7 M urea). About 0.4 A260 units of tRNA transcript (containing 32P around 18 000 dpm) and 2 µg of the purified TruB protein in 40 µl buffer A were incubated at 55°C for 1 h. The RNA was extracted with phenol–chloroform and then recovered by ethanol precipitation. The RNA pellet was dissolved in 3 µl of 50 mM sodium acetate (pH 5.0) and digested with 2.5 U of nuclease P1 (Wako Pure Chemicals). The sample was separated by two dimensional thin layer chromatography (2D-TLC) as described previously (54). The 32P-labeled nucleotides were monitored with a Fuji Photo Film BAS2000 imaging analyzer.

Construction of Δ truB (ΔTTHA0217) strain

The TTHA0217 gene was disrupted by replacement with the highly thermostable kanamycin nucleotidyltransferase (HTK) gene (55,56). The plasmid vector containing the TTHA0217 region disrupted by the HTK gene was purchased from RIKEN Biological Resource Center (Tsukuba, Japan) (52). Thermus thermophilus cells in late-log phase were transformed by the vector according to the report of Hashimoto et al. (57) and mutant colonies were selected on a plate containing 500 µg/ml kanamycin at 70°C. The genomic DNA from each colony was isolated, analyzed by PCR and then the sequence of the replaced region was determined on ABI PRISM 310 DNA sequencers.

Purification of native tRNAMetf1 by solid-phase DNA probe

The 3′-Biotinylated DNA oligomer (5′-GGG TTA TGA GCC CGA CGA GCT ACC –biotin-3′) was used as a hybridization probe. The probe sequence is complementary from C41 to G19 of T. thermophilus tRNAMetf1. Before solid phase DNA probe column chromatography, tRNAMetf1 fraction was prepared by BD-cellulose column chromatography as reported previously (58). Purification of tRNAMetf1 by solid-phase DNA probe was performed as described in our recent reports according to the suggestions of Dr Yokogawa (Gifu University) (59–61). The eluted tRNAMetf1 was further purified by 10% PAGE (7 M urea).

Nucleosides analysis by HPLC

Nucleoside analysis by HPLC was performed according to our recent report (60).

Construction of T. thermophilus truB mutant strain

Site-directed mutagenesis was performed using the Quick change mutagenesis kit (Stratagene). Schemes of construction of plasmid vectors for gene replacement of the truB mutant genes are shown in Supplementary Figure S3. The homologous recombination was performed as described above. The genomic DNA was isolated, analyzed by PCR and then the sequence of the replaced region was determined on ABI PRISM 310 DNA sequencers.

Western blotting analysis

Customized rabbit anti-T. thermophilus TruB serum was prepared by Oriental Kobo Co., Ltd, Japan. The polyclonal antibody fraction was partially purified using the Econo-pac serum IgG purification kit (Bio-Rad). Cultured cells were directly resuspended in SDS–PAGE sample buffer and then disrupted. The samples were boiled and immediately loaded onto a 15% SDS–polyacrylamide gel. Electro-blotting to a nitrocellulose membrane (Nitro Bind membrane code EP2HY315F5, pore size 0.45 µm, Osmonics) was performed using a semi-dry blotting system (NA-1515B, Nippon Eido) according to the manufacturer’s instructions. TruB protein was detected using Alexa Fluor 488 anti-rabbit IgG (Invitrogen) as a secondary antibody and visualized using a Typhoon model 9410 (GE healthcare).

Preparation of tRNA modification enzymes

Thermus thermophilus tRNA (Gm18) methyltransferase (TrmH) and Aquifex aeolicus tRNA (m7G46) methyltransferase (TrmB) were purified according to our previous reports (41,62). Thermus thermophilus tRNA (m1A58) methyltransferase (TrmI) (63) was a gift from Masayuki Minoji (Ehime University). Escherichia coli tRNA (m5U54) methyltransferase (TrmA) (47–49) was expressed in E. coli and purified as follows. TrmA coding region was amplified by PCR from E. coli BL21 (DE3) genomic DNA using the following primers: EcTrmAN, 5′-CCC CCA TAT GAC CCC CGA ACA CCT TCC ACC-3′; EcTrmAC, 5′-CCC CGG ATC CTT ACT TCG CGG TCA GTA ATA CGC CGC ACT CCA TGT GGT GC-3′. Underlining shows restriction enzyme sites (Nde I and Bam HI). Double underlining shows a mutation site for disruption of the Nde I site, which exists in the original E. coli genomic DNA. This mutation does not change the amino acid sequence of TrmA. The amplified DNA was digested with Nde I and Bam HI, and ligated into the multi-cloning linker of pET30a E. coli expression vector. The expression of TrmA protein in E. coli BL21 (DE3) Rosetta 2 strain was performed according to the manufacturer’s manual. Cells (4.5g) were suspended in 20 ml of buffer C [10 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.5 mM DTT and 10% glycerol] and then disrupted with an ultrasonic disruptor model UD-200 (Tomy, Japan). The cell debris was removed by centrifugation at 8000g for 20 min and then ammonium sulfate was added to the supernatant fraction (final concentration, 1.7 M). The sample was further centrifuged at 24 000g for 20 min. The supernatant fraction was passed through a Steradisc 25 filter (0.2 µm, Kurabo) and then loaded onto a HiPrep Phenyl FF low sub 16/10 column (GE Healthcare). A liner gradient was developed from 1.7 M to 0 M ammonium sulfate in buffer C. TrmA fractions were assessed by 15% SDS–PAGE, combined, dialyzed against buffer A and concentrated with Centriprep YM-10 centrifugal filter devices (Millipore). Glycerol was added to the purified protein to a final concentration of 50% v/v and stored at −30°C.

In vitro tRNA modification enzyme assay

To estimate the content of unmodified nucleotides in tRNA, tRNA mixtures from the wild-type and ΔtruB strains were methylated with purified tRNA modification enzymes. The tRNA mixtures were prepared from the cells cultured at 50°C. About 0.2 A260 units of tRNA mixture, enzyme (45.0 pmol TrmH, 28.6 pmol TrmB or 17.5 pmol TrmI) and 19 µM 14C-AdoMet were incubated in 40 µl buffer A at 55°C over night. In the case of TrmA (57 pmol), the incubation temperature was 37°C. The 14C-methyl group incorporation was monitored by a liquid scintillation counter using a filter assay. For the time-course assay, T. thermophilus class I tRNA from the ΔtruB strain with the Ψ55 modification was prepared as follows. About 1.63 A260 units of class I tRNA from the ΔtruB strain was incubated with 1.0 nmol wild-type TruB in 100 µl buffer A at 55°C for 3 h. The sample was extracted with phenol and the RNA was recovered by ethanol precipitation. We checked the purity of the recovered transcript by 10% PAGE (7 M urea). The time-course assay was performed as follows: 0.1 A260 units of class I tRNA fraction with or without Ψ55 modification, 26 µM 3H-AdoMet and 45.0 pmol TrmH were incubated in 30 µl buffer A at 55°C.

Melting profile analyses of class I tRNA

Class I tRNA fractions from the wild type and ΔtruB strains cultured at 50°C and 70°C were prepared as described above. Before the melting point measurement the tRNA fraction was annealed in buffer D [50 mM Tris–HCl (pH7.5), 5 mM MgCl2, 100 mM NaCl] from 80°C to 40°C for 60 min and then the melting curve was recorded on a spectrophotometer, UV-1650PC (Shimadzu). The temperature was increased from 40 to 95°C over 55 min. The melting profiles were obtained by averaging two scans. The melting temperatures were calculated from first derivative plots.

Translation activity analyses by 35S-Met incorporation

The wild-type and ΔtruB strains were cultured at 50 or 70°C. When the cell density (A600 nm) had reached 0.6, the culture medium (60 ml) was supplemented with 12.0 MBq of 35S-Met. The sample (15 ml each) was taken at 0, 15, 30 and 60 min. The cells were collected by centrifugation at 3500g for 5 min. The cells were washed with 5 ml of buffer A and collected by centrifugation at 3500g for 5 min. The cells were resuspended in 120 µl of buffer A after which 120 µl of SDS loading buffer [100 mM Tris–HCl (pH 6.8), 200 mM dithiothreitol, 2.5% SDS, 0.2% bromophenol blue, 20% glycerol] was added. The sample was boiled for 10 min and centrifuged at 21 500g for 10 min. The supernatants were analyzed by 15% SDS–PAGE. The gels were stained with Coomassie brilliant blue and 35S-Met incorporation was monitored with a Fuji Photo Film BAS2000 imaging analyzer.

RESULTS

The Ψ55 modification is absent in the purified tRNAMetf1 from the ΔTTHA0217 strain

As shown in Figure 1A, the RNA sequence of T. thermophilus tRNAMetf1 has been reported and this tRNA has only one Ψ modification at position 55 (58). In order to check whether the TTHA0217 gene is truB, we purified this tRNA by solid phase DNA probe column chromatography (59–61). Thermus thermophilus has two initiator tRNAMet species, tRNAMetf1 and tRNAMetf2 (58). Because the only difference between these initiator tRNAMet species is one base pair in their T-stem, separation of these tRNA species by DNA probe proved impossible. Therefore, we initially separated two initiator tRNAMet species by BD-cellulose column chromatography as described (58) and then the tRNAMetf1 rich fraction was used for the solid phase DNA probe column chromatography. We successfully purified tRNAMetf1 from the wild-type and ΔTTHA0217 strains (Figure 1B). The modified nucleosides of these tRNA species were analyzed by HPLC C18 reverse phase column chromatography (Figure 1C). The Ψ nucleoside was eluted at 6.7 ml on our HPLC system. As shown in Figure 1C (upper panel), the Ψ nucleoside was clearly observed in the wild-type strain sample. In contrast, the peak of Ψ nucleoside was missing in the tRNAMetf1 from the ΔTTHA0217 strain (Figure 1C, lower panel), demonstrating that the TTHA0217 gene is T. thermophilus truB. Hereafter, we describe ΔTTHA0217 as ΔtruB.

Figure 1.

Figure 1.

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The Ψ55 modification is absent in the purified tRNAMetf1 from the ΔTTHA0217 strain. (A) Cloverleaf structure of T. thermophilus tRNAMetf1. This tRNA has a single Ψ modification at position 55. The complimentary region to the DNA probe is illustrated. (B) Purified tRNAMetf1 molecules from the wild-type and ΔTTHA0217 strains were analyzed by 10% PAGE (7 M urea). The gel was stained with toluidine blue. (C) The modified nucleosides in tRNAMetf1 from the wild type (upper) and the ΔTTHA0217 (lower) cells cultured at 50°C were compared.

Growth retardation of the ΔtruB strain at low temperatures

We examined the growth phenotype of the ΔtruB strain at various temperatures. Figure 2 shows the results of liquid cultures at 50°C, 60°C, 70°C and 80°C. The wild type strain can live at a wide range of temperature (50–80°C). In contrast and to our surprise, severe growth retardation of the ΔtruB strain was observed at 50°C (Figure 2A). At 60°C, the growth retardation was mitigated (Figure 2B). At 70°C, the wild-type and ΔtruB strains exhibited similar growth (Figure 2C). At 80°C, a curious growth phenotype of the ΔtruB strain was observed: the ΔtruB strain did not have a lag-phase and showed an immediate exponential growth, which was slower than that of the wild-type strain (Figure 2D). It should be mentioned that the pre-cultures were performed at 70°C. We repeated the experiments and confirmed that growth phenotypes were reproducibly observed at all temperatures. In general, the Ψ55 modification contributes to stabilization of the L-shaped tRNA structure. Therefore, growth retardation at 80°C seemed to be reasonable although we could not explain the reason why the ΔtruB strain did not show a lag-phase like the wild-type strain. We focused on unexpected growth retardation at low temperatures (<60°C). At low temperatures, tRNA structure is more stable compared to that at high temperatures. Furthermore, in general, lack of single modification in the three-dimensional core of tRNA does not cause any effect on cell growth at low temperatures (64). Even in the case of T. thermophilus, only three modifications [m7G46 (60), m5s2U54 (65) and m1A58 (63)] have been reported to affect cell growth and then only at high temperatures (especially 80°C). In the recent study, we reported that hypo-modifications in tRNA at high temperatures caused degradation of some tRNA species (60). However tRNA degradation only at low temperatures could not be assumed. Thus, this unexpected growth retardation at low temperatures is not explainable by loss of structural stability of tRNA through lack of the Ψ55 modification.

Figure 2.

Figure 2.

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Growth phenotypes of the wild-type and ΔtruB strains. The growth curves of the wild-type and ΔtruB strains in liquid cultures at 50°C (A), 60°C (B), 70°C (C) and 80°C (D) are shown.

Construction of T. thermophilus strains, which express mutant TruB proteins, for detection of RNA chaperone activity

Initially, we considered that lack of the RNA chaperone activity of TruB might cause the growth retardation at low temperatures if the RNA chaperone activity was genuine. The RNA chaperone activity of TruB protein in assisting the correct folding of tRNA was proposed by a competitive growth experiment with E. coli ΔtruB strain and the wild-type strain (37). We considered that the RNA chaperone activity of TruB might be required only at low temperatures in the case of T. thermophilus because tRNA has structural softness (flexibility) at high temperatures. To verify this idea, we constructed T. thermophilus strains, which express mutant TruB proteins. Fortunately, the catalytic mechanism of TruB has been well studied (28–36) and the catalytic center has been verified (33,34). Based on amino acid sequence alignment (Supplementary Figure S1), we expected the aspartic acid at position 37 (D37), which corresponds to D48 in E. coli TruB, to be the catalytic center. We substituted D37 by alanine and cysteine individually because it was reported that these substitutions of the catalytic center produced severe loss of enzymatic activity (36). We purified recombinant D37A and D37C mutant proteins which were expressed in E. coli cells (Figure 3A). Ψ formation activities were checked by 2D-TLC. Briefly, T. thermophilus tRNAThr (GGT) was transcribed with T7 RNA polymerase and α-32P-UTP. The transcript was treated with the TruB wild-type, D37A, or D37C protein at 55°C for 1 h and then digested with nuclease P1 completely. The resultant nucleotides were separated on 2D-TLC and analyzed by autoradiography. As shown in Figure 3B, 32P-pΨ was clearly observed in the wild-type sample. In contrast, 32P-pΨ was not detected in the D37A sample (Figure 3C), demonstrating a near-completely loss of activity in the D37A mutant protein. Furthermore, a faint spot of 32P-pΨ was observed in the D37C sample (Figure 3D), showing that the D37C mutant protein has very weak Ψ formation activity as in the case of E. coli TruB (36). It should be mentioned that Figure 3 does not represent the relative initial velocities: in order to demonstrate the weak activity of the D37C mutant enzyme, incubation was prolonged for 1 h. The activity of the D37C mutant enzyme is ∼1% of that of the wild-type enzyme (data not shown). Thus, we successfully prepared mutant truB genes. We constructed the gene replacement vectors as shown in Supplementary Figure S3 and performed homologous recombination. Thermus thermophilus mutant strains were isolated and their DNA sequences were checked (data not shown). Expression of the mutant TruB proteins in T. thermophilus cells was confirmed by western blotting analysis (Figure 3E). The TruB mutant proteins as well as the wild-type protein were clearly detected in the S-100 fractions. In contrast, the band corresponding to the TruB protein is absent in the ΔtruB strain sample. Thus, we confirmed the expression of TruB D37A and D37C proteins in the T. thermophilus mutant strains. Ψ contents in tRNA from the wild-type, ΔtruB, D37A and D37C strains were checked by the HPLC nucleoside analysis of class I tRNA fractions (Figure 3F) because removal of rRNA fragments from total tRNA fraction was difficult. The wild-type, ΔtruB, D37A and D37C strains were cultured at 60°C. Total tRNA fraction from each strain was prepared by phenol extraction and then the class I tRNA fraction was excised by 10% PAGE (7 M urea) as shown in the insets of Figure 3F. Each class I tRNA was then completely digested with phosphodiesterase, bacterial alkaline phosphatase and RNase A. In the class I tRNA from the wild-type strain, Ψ was clearly observed because five types of tRNA Ψ synthases (TruA, TruB, RsuA, RluA and TruD) probably exist in the cells. In contrast, in the class I tRNA from the ΔtruB strain, the content of Ψ was dramatically decreased, consistent with deletion of truB. A similar result was obtained from the D37A mutant strain sample, suggesting that Ψ55 in tRNA is absent even though mutant TruB D37A protein is expressed. Furthermore, Ψ content in the class I tRNA from the D37C mutant strain was slightly increased relative to the D37A mutant, consistent with the weak Ψ55 formation activity of TruB D37C mutant protein. Thus, we confirmed that T. thermophilus TruB D37A and D37C mutant strains were successfully constructed.

Figure 3.

Figure 3.

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Enzymatic activities of the TruB wild-type, D37C and D37A proteins. (A) The TruB wild-type, D37A and D37C proteins were purified to near-homogeneity on 15% SDS–PAGE analysis. The gel was stained with Coomassie brilliant blue. The Ψ55 formation activities of the wild-type (B), D37A (C) and D37C (D) proteins were checked by 2D-TLC using 32P-internal labeled tRNAThr transcript as described in the text. (E) The presence of the TruB wild-type, D37A and D37C proteins in the cell extracts were checked by western blot analysis. In contrast, the band corresponding to TruB was absent in the ΔtruB strain sample. (F) Nucleosides in 0.2 A260 units of class I tRNA fractions from the wild-type and ΔtruB cells cultured at 60°C were analyzed by HPLC C18 reverse phase column chromatography. The class I tRNA fractions were analyzed by 10% PAGE (7 M urea) (Insets). The Ψ nucleoside was eluted at 6.7 ml. Elution points of the labeled nucleosides were confirmed by standard markers or enzyme formation as described in our recent report (60).

RNA chaperone activity of TruB protein might exist, but the growth retardation at low temperatures could not be explainable by the RNA chaperone activity

In order to clarify whether the RNA chaperone activity of TruB really exists and has positive effects on cell growth, we investigated the growth phenotype of the wild-type, ΔtruB, D37A and D37C strains at 50°C (Figure 4A). To our disappointment, the growth of the D37A strain was not perfectly recovered but was slightly faster than that of the ΔtruB strain. In contrast, the growth of the D37C strain was obviously faster than that of the D37A and ΔtruB strains. Furthermore, the wild-type strain grew faster than any mutant strain. Thus, these experimental results revealed that the growth retardation of the ΔtruB strain at 50°C was mainly caused by lack of the Ψ55 modification in tRNA although our experimental results did not contradict the RNA chaperone hypothesis. Thus, the growth retardation was not explainable by the RNA chaperone activity. We further analyzed the growth phenotypes at 60°C, 70°C and 80°C (Figure 4B–D). At 60°C, recovery of growth by expression of the D37A and D37C mutant proteins was still observed however the effect was weaker than that at 50°C (Figure 4B). At 70°C, all strains showed similar growth (Figure 4C). At 80°C, unexpectedly, both D37A and D37C mutant strains grew considerably faster than the ΔtruB strain without the presence of a lag-phase. These phenomena might be caused by the RNA chaperone activity, which functions in exponential growth at high temperatures.

Figure 4.

Figure 4.

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Effect of the TruB mutant proteins on cell growth was tested. The growth curves of the wild-type, ΔtruB, D37A and D37C strains were compared at 50°C (A), 60°C (B), 70°C (C) and 80°C (D).

Abnormal enhancement of other tRNA modifications in the ΔtruB strain at 50°C

During the course of the study, we investigated the T. thermophilus trmB disruption strain (60). The gene, trmB encodes tRNA (m7G46) methyltransferase (60,62,66). In a previous study, we found that the lack of m7G46 modification causes hypo-modifications of other modified nucleotides such as Gm18 and m1G37 (60). Thus, m7G46 modification has positive effects on other modifications, suggesting that there is a tRNA modification network(s). We considered that Ψ55 might have effects on other tRNA modifications. To confirm this idea, we performed nucleoside analysis of class I tRNA from the wild-type and ΔtruB strains cultured at 50°C and 70°C. The class I tRNA fractions were prepared as shown in Figure 5A. Nucleosides in these class I tRNA fractions were analyzed (Figure 5B and C). Surprisingly, the amount of several modified nucleotides such as m1A, Gm + m1G and m5s2U increased in the ΔtruB strain cultured at 50°C as compared to that in the wild-type sample (Figure 5B). It is known that the amount of these modifications (m1A, Gm and m5s2U) in T. thermophilus tRNA increase with culture temperature (58,60,67). In fact, these modifications in the class I tRNA from the wild-type strain cultured at 70°C clearly increased as compared to those at 50°C (Figure 5B and C, upper panels). However these nucleosides in the class I tRNA of the ΔtruB strain cultured at 50°C were abnormally increased (Figure 5B, lower panel).

Figure 5.

Figure 5.

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Nucleoside analysis of class I tRNA fractions from the wild-type and ΔtruB cells cultured at 50°C and 70°C. (A) The class I tRNA fractions (0.05 A260 units) were analyzed by 10% PAGE (7M urea). The gel was stained with toluidine blue. The modified nucleosides in the class I tRNA fractions from the cells cultured at 50°C (B) and 70°C (C) were analyzed by HPLC C18 reverse phase column chromatography. The ratio of modified nucleoside was calculated from the peak area: the peak area in the wild-type sample at 50°C was expressed as 100%.

Modification assay for tRNA methyltransferases

In order to estimate the modified nucleoside contents in tRNA correctly, we prepared four tRNA modification enzymes, TrmH, TrmB, TrmA and TrmI (Figure 6A). TrmH, TrmB, TrmA and TrmI are tRNA (Gm18) methyltransferase (40–42), tRNA (m7G46) methyltransferase (60,62,66), AdoMet-dependent tRNA (m5U54) methyltransferase (47–49) and tRNA (m1A58) methyltransferase (63), respectively. It should be mentioned that the m5U54 modification in T. thermophilus is generated by the N5, N10-methylene tetrahydrofolate-dependent enzyme (TrmFO) (50,51). However N5, N10-methylene tetrahydrofolate is unstable and radioisotope labeled compound is not commercially available. Therefore we used E. coli TrmA instead of TrmFO. The tRNA mixtures from the wild-type and ΔtruB strains cultured at 50°C were methylated with 14C-AdoMet by addition of these individual enzymes for 1 h, 3 h and over-night. Because the 14C-methyl group incorporation was not changed for 3 h and over-night incubations, we judged that the tRNA mixtures were near-completely methylated (Figure 6B). As shown in Figure 6B, the Gm18 and m1A58 content in the wild-type sample was clearly lower than that seen in the ΔtruB strain sample, consistent with the HPLC nucleoside analysis. These results suggest that the Ψ55 modification has a negative effect on the TrmH and TrmI reactions. The m7G46 modification was near-completed in both the wild-type and ΔtruB strain samples although the content in the ΔtruB strain was slightly more than that in the wild-type sample, suggesting that the presence of the Ψ55 modification does not affect the TrmB reaction significantly. To our surprise, a considerable amount of U54 in both the wild-type and ΔtruB strains remained as unmodified U54 (or s2U54), demonstrating that TrmFO works effectively at high temperatures. Thus, we confirmed that the Ψ55 modification does not significantly affect methylation of U54. It should be mentioned that nucleoside analysis in Figure 5B clearly showed an increase of m5s2U in the ΔtruB strain cultured at 50°C. Taking these experimental results together, we conclude that the presence of the Ψ55 modification has a negative effect on formation of the m5s2U54 from the m5U54 although the Ψ55 modification does not have a significant effect on the m5U54 modification at least at 50°C. The sulfur-transfer reaction in m5s2U54 formation is very complicated as reported by Shigi with at least, four proteins (TtuA, TtuB, TtuC and IscS) being involved in the reaction (65,67,68). Therefore we could not investigate the effect of the Ψ55 modification on the sulfur-transfer reaction enzymatically. However our current experimental results suggest two hypothetical schemes. One is that the Ψ55 modification has a direct negative effect on these sulfur-transfer proteins (or complex). The other is that the Ψ55 modification has a negative effect only on the m1A58 formation and enhanced m1A58 modification through a lack of the Ψ55 modification inducing the sulfur transfer reaction since the m1A58 modification has been reported to be a positive determinant for the sulfur-transfer reaction (67).

Figure 6.

Figure 6.

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Modification assay with tRNA methyltransferases. (A) T. thermophilus tRNAPhe is depicted in the cloverleaf structure. Modification sites (Gm18, m7G46, m5U54 and m1A58) are shown in the figure. The corresponding modification enzymes (TrmH, TrmB, TrmA and TrmI) on 15% SDS–PAGE are shown. Each gel was stained with Coomassie brilliant blue. (B) Mixtures of tRNA from the wild-type and ΔtruB cells cultured at 50°C were prepared and then served as substrate for tRNA methyltransferases as described in ‘Materials and Methods' section. (C) Thermus thermophilus class I tRNA from ΔtruB strain were modified with TruB for 3 h at 55°C. Then methyl group acceptance activities with and without Ψ55 modification in reactions catalyzed by TrmH were compared.

Next, we performed a time-dependent assay with unmodified and Ψ55 modified class I tRNA from the ΔtruB strain. The class I tRNA from the ΔtruB strain was modified with TruB protein for 3 h and then used as the substrate for TrmH. As shown in Figure 6C, for TrmH, unmodified class I tRNA was modified faster than the class I tRNA with Ψ55. Thus, in vitro TrmH reaction is consistent with the in vivo modification. The negative effect of the Ψ55 modification on Gm18 modification is explainable without considering other protein (or RNA) factors. The presence of the Ψ55 modification brings rigidity to the local structure of tRNA (5). This rigidity probably decreases the speed of the induced fit process of TrmH at least at 55°C (46).

Abnormal high melting temperature of class I tRNA from the ΔtruB strain

In the absence of the Ψ55 modification, the amounts of Gm18, m1A58 and m5s2U54 modifications were abnormally increased. In this case the melting temperature of tRNA from the ΔtruB strain would be expected to be increased. We therefore analyzed the melting profiles of class I tRNAs from the wild-type and ΔtruB strains cultured at 50°C and 70°C (Figure 7A and B). The melting temperature of tRNA from the ΔtruB strain cultured at 50°C was much higher than that of the wild-type sample (Figure 7A and C). This abnormally high melting temperature suggests that the tRNA structure may be too rigid to work at 50°C, because the m5s2U54 modification in tRNAPhe depresses the poly (U) dependent-poly (Phe) translational activity at 50°C in the case of T. thermophilus (69). In contrast, tRNAs from the wild-type and ΔtruB strains cultured at 70°C showed similar melting profiles, consistent with the growth phenotypes (Figure 2C) and nucleoside analysis (Figure 5A).

Figure 7.

Figure 7.

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Melting profiles of the class I tRNA from the wild-type and ΔtruB cells cultured at 50°C (A) and 70°C (B). The profiles >90°C were perturbed by bubbles generated in the samples with increased temperature. The calculated melting temperatures are summarized in (C).

35S-Met incorporation of the ΔtruB strain is inferior to that of the wild-type strain at 50°C and 70°C

In order to verify whether the protein synthesis activity of the ΔtruB strain is depressed at 50°C, we investigated the 35S-Met incorporation (Figure 8). The wild-type and ΔtruB strains were cultured at 50°C or 70°C. When the cell density (A600 nm) had reached 0.6, 35S-Met was directly supplemented into the medium. As shown in Figure 8, 35S-Met incorporation into the wild-type cells was clearly observed at both 50°C and 70°C. In contrast, 35S-Met incorporation of the ΔtruB strain was hardly detectable at 50°C. Although 35S-Met incorporation of the ΔtruB strain was observed at 70°C, it was considerably inferior to that of the wild-type strain. Thus, these results clearly showed that the protein synthesis activity of the ΔtruB strain is inferior to that of the wild-type strain at both 50°C and 70°C.

Figure 8.

Figure 8.

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Translation activity analysis by 35S-Met incorporation. The wild-type and ΔtruB cells were cultured at 50°C (left) and 70°C (right). At 0 min point, 35S-Met was supplemented into the medium. The cells were collected at 0, 15, 30 and 60 min. The proteins were analyzed by 15% SDS–PAGE (upper). The gel was stained with Coomassie brilliant blue. The lower panels show the autoradiogram of the gel.

These experiments brought us additional information. In the case of the wild-type strain, synthesized protein species were different at 50°C and 70°C (Figure 8). At 50°C, small proteins were abundantly synthesized. In fact, accumulation of the small proteins at 50°C was observed in the wild-type sample (Figure 8, upper panel). These bands were corresponding to the sizes of the cold-shock proteins in T. thermophilus. Thermus thermophilus has at least two cold-shock proteins (ttCsp1 and ttCsp2) (70). The expected sizes of ttCsp1 (TTHA0175) and ttCsp2 (TTHA0359) are 8184 and 7768 Da, respectively. In contrast, these bands were not observed in the ΔtruB strain sample. Thus, the depression of the protein synthesis activity in the ΔtruB strain causes deficit of the cold-shock proteins at 50°C. Probably, the deficit of the cold-shock proteins blunts the cell responses in metabolism, transcription, translation and protein folding to low temperatures (70) and causes severe growth defect at 50°C.

DISCUSSION

In this study, we investigated a truB gene disruptant mutant of T. thermophilus, an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at low temperatures (especially 50°C). Some tRNA modifications such as Gm18, m5s2U54 and m1A58 were abnormally enhanced in class I tRNA from the ΔtruB strain cultured at 50°C. These enhanced modifications produce an unwanted increase in the melting temperature of tRNA. In fact, the protein synthesis activity of the ΔtruB strain was clearly inferior to that of the wild-type strain at both 50°C and 70°C. Furthermore, the bands corresponding to the cold-shock proteins were absence in the ΔtruB strain sample cultured at 50°C. This deficit of the cold-shock proteins in the ΔtruB cells probably causes the severe growth defect at 50°C. At least at 50°C, the Ψ55 modification controls the content of other modifications in tRNA. This idea was confirmed by nucleoside analysis of modified tRNA in vivo and in an in vitro modification assay with tRNA modification enzymes. Thus, the Ψ55 modification is required for low-temperature adaptation for the organism. For a long time, modified nucleotides in the 3D-core of tRNA have been mainly considered as tRNA stabilization factors. However our current study provides a new concept, namely that a decrease of amounts of modified nucleotides in the 3D-core of tRNA brings structural softness (flexibility) to tRNA in some cases (for example, at low temperatures for T. thermophilus) and functions as an environment adaptation factor. In this system, the Ψ55 modification controls the tRNA modification network.

Based on our previous (60) and current studies and research by others (63,67), the network of modified nucleotides and tRNA modification enzymes in T. thermophilus can be drawn as shown in Figure 9. It should be mentioned that each modification enzyme can act on unmodified tRNA transcript. This figure shows positive and negative effects of the modified nucleotides on other modification enzymes. Of course, the modified nucleotide itself acts as a competitive inhibitor of the corresponding modification enzyme. For example, increase of Gm18 content in tRNA inhibits TrmH activity (46). Furthermore, this network is observed in extreme thermophilic eubacterium T. thermophilus, which can grow at 50–80°C. In the network, two modified nucleotides, Ψ55 and m7G46 work as key factors. As described in this article, the presence of the Ψ55 modification has a negative effect on TrmH activity in vivo and in vitro. Although sulfur-transfer to m5U54 increases in the ΔtruB strain cells cultured at 50°C, this phenomenon is not confirmed by in vitro assay. Therefore, this may be indirectly induced by increase in m1A58 content (63,67). The negative effect of the Ψ55 modification on m1A58 modification was confirmed in vivo. To clarify the precise modification mechanism by TrmI, further study will be necessary. In contrast, the presence of m7G46 modification accelerates modification velocities of Gm18 by TrmH and m1G37 by TrmD in vivo and in vitro (60). The positive effect of the m7G46 modification on TrmI activity was confirmed only in vitro, suggesting that a sufficient amount of the TrmI protein may exist in the T. thermophilus cells (60). In the network, the m5s2U54 modification mainly contributes to increase of melting temperature (5,58,67). Although all protein factors for the m5s2U54 modification have not been identified (65,68), understanding of the m5s2U54 formation pathway will be necessary to elucidate the all of the temperature-dependent tRNA adaptation system of thermophiles. In Figure 9, not all common modifications in the 3D-core are depicted. For example, D20 modifications in tRNAMetf and tRNAPhe from T. thermophilus have been reported (2). In the case of E. coli, the D modifications in tRNA are produced by three dihydrouridine synthases (Dus) (71) however only one gene could be found in T. thermophilus genome. Therefore, experimental identification of the dihydrouridine synthase gene in T. thermophilus is necessary. In general, the D modification confers flexibility to the local structure of RNA (72). Furthermore, in the case of yeast, the Gm18 modification by Trm3 is required for the D20 modification by Dus2 (73–75). In the future study, role of the D modification(s) should be investigated in T. thermophilus tRNA modification network.

Figure 9.

Figure 9.

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Modified nucleotide and modification enzyme network in T. thermophilus tRNA. (A) The modification positions are depicted in the cloverleaf structure of typical tRNA. The conserved nucleotides are shown by A, U, G and C. Py and Pu are abbreviations for pyrimidine and purine, respectively. Broken lines are tertiary base pairs. Numbers show positions of the modifications. (B) This figure shows the positive or negative effect of modified nucleotide on other modifications. Nucleotides and positions are boxed. The modification enzyme which catalyzes each reaction is shown for each pathway. This network maintains an adequate balance of tRNA modifications and responds to environmental changes (for example, culture temperature).

In the current study, we found that expression of D37A mutant protein clearly produced recovery of the growth speed at 80°C (Figure 4D). At 50°C, a slight recovery in the growth speed was also observed (Figure 4A). Because we constructed the mutant strain by homologous recombination, the mutant TruB gene existed as only one copy in the genome. If we supplied the mutant protein into the cell by plasmid vectors, the recovery at 50°C would be expected to be enhanced. Our experimental results do not contradict the hypothesis by Gutgsell et al. (37). However there are two other possibilities. One is that the TruB protein binds to unidentified protein(s) and this complex has another function at 80°C. Because TruB protein itself may affect other proteins on the precursor tRNA, we cannot neglect this possibility. The other is that TruB protein binds to tRNA and protects from cleavages by nucleases. Although the amount of tRNA is much than that of the TruB, this protection should be considered. If the TruB has the RNA chaperone activity, it suggests one idea: other 3D-core modification enzymes may have a RNA chaperon activity. To clarify the idea, further studies will be necessary. The D37A and D37C mutants as well as the ΔtruB strain do not show a lag-phase in the culture at 80°C. This fact suggests that the Ψ55 modification works at a lag-phase in the modified nucleotide and modification enzyme network. Majority of the basic modifications in tRNA seemed to be taking place in a lag-phase.

The Ψ55 modification was found in A. aeolicus tRNACys (59) and T. maritima has a TruB protein (27,28). Furthermore truB genes are found in almost all eubacterial genomes (23,24). These facts suggest that the Ψ55 modification and TruB protein existed in the common ancestor of eubacteria. Because the common ancestor probably lived in a high-temperature environment, the Ψ55 modification probably functioned on tRNA stabilization. According to cooling of the Earth, thermophiles should adapt to low temperatures for survival. During this process, the Ψ55 modification might have obtained a new function, which controls other modifications. In mesophiles, the Ψ55 modification may have been changed to a stress resistance factor (25,26,37,39).

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

Supplementary Data
supp_39_6_2304__index.html (1.3KB, html)

FUNDING

Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientist (20-4827 to C.T. and 21-10011 to A.O.); a Grant-in-aid (20034041 to H.H.) for Science Research on Priority Areas; Grant-in-aid (19350087 to H.H.) for Science Research from the Ministry of Education, Science, Sports and Culture of Japan. Funding for open access charge: JSPS Research Fellowships for Young Scientist (20-4827 to C.T. and 21-10011 to A.O.); a Grant-in-aid (20034041 to H.H.) for Science Research on Priority Areas; Grant-in-aid (19350087 to H.H.) for Science Research; Grant-in-aid (21770145 to A.H.) for Young Scientists from the JSPS.

Conflict of interest statement. None declared.

ACKNOWLEDGEMENTS

We thank Prof. Yaeta Endo (Ehime University) for encouraging our study.

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Supplementary Data
supp_39_6_2304__index.html (1.3KB, html)
supp_gkq1180_SFig_2.ps (583.9KB, ps)
supp_gkq1180_SF_1.ps (1.4MB, ps)
supp_gkq1180_SF_3.ps (433.2KB, ps)
supp_gkq1180_Supplementary_file.doc (33.5KB, doc)

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