N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

Abstract

N(7)-methylguanine at position 46 (m(7)G46) in tRNA is produced by tRNA (m(7)G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (DeltatrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the DeltatrmB strain and the lack of the m(7)G46 modification in tRNA(Phe) were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the DeltatrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m(7)G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m(1)G37, suggesting that the m(7)G46 positively affects their formations. Although the lack of the m(7)G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA(Phe), they cause a decrease in melting temperature of class I tRNA and degradation of tRNA(Phe) and tRNA(Ile). (35)S-Met incorporation into proteins revealed that protein synthesis in DeltatrmB cells is depressed above 70 degrees C. At 80 degrees C, the DeltatrmB strain exhibits a severe growth defect. Thus, the m(7)G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m(7)G46 modification supports introduction of other modifications.

Figure 1.

Absence of m⁷G formation activity and lack of m⁷G nucleoside in extract and tRNA from the ΔTTHA1619 strain. (A) yeast tRNA^Phe transcript, [methyl-¹⁴C]-AdoMet and S-100 fraction of the wild-type (left) or ΔTTHA1619 (right) strain were incubated at 60°C for 1 h, and ¹⁴C-methylated nucleotides were analyzed by 2D-TLC. Positions of standard markers (pA, pG, pC and pU) are enclosed by dotted circles. (B) nucleoside analyses of the class I tRNA fractions from the wild-type (upper) and ΔTTHA1619 (lower) strains. 0.03 A₂₆₀ units of the purified class I tRNA fractions were analyzed by 10% PAGE (7 M urea) and the gel was stained with toluidine blue (insets).

Figure 2.

Sequence analyses of purified tRNA^Phe from the wild-type and ΔTTHA1619 strains by Kuchino’s post labeling method. (A) Nucleotide sequence of T. thermophilus tRNA^Phe is depicted as a cloverleaf structure. The 3′-biotin DNA probe is illustrated. The m¹A58 modification was identified in this work. (B) 0.01 A₂₆₀ units of purified tRNA^Phe from the wild-type (left) and ΔTTHA1619 (right) strains were analyzed by 10% PAGE (7 M urea). The gel was stained by toluidine blue. The purified tRNA^Phe from the wild-type (C) and ΔTTHA1619 (G) strains was partially cleaved by formamide. Then the 5′-end of each fragment was labeled with γ-³²P-ATP and T4 polynucleotide kinase. The RNA fragments were separated by 15% PAGE (7M urea). Numbers correspond to the nucleotide positions in tRNA^Phe. The tRNA^Phe fragments of the wild-type (D) and ΔTTHA1619 (H) strains were cut off from the gels in panels C and G, respectively. The fragments were digested with nuclease P1 and their 5′-nt were analyzed by TLC. In panels D and H, nucleotides at positions from 30 to 48 are shown. Positions of standard markers (pA, pG, pC and pU) are indicated by arrows at the right side of the thin layer plates. (E, I) Modified nucleotides of panels D and H were analyzed by 2D-TLC. The arrows indicate spots of modified nucleotides. (F) TLC patterns of the all modified nucleotides identified in tRNA^Phe from the wild-type strain are shown.

Figure 3.

Growth phenotypes of the wild-type and ΔtrmB strains. (A) The wild-type and ΔtrmB strains were serially diluted, spotted onto plates containing rich medium, and incubated at the temperatures indicated. Incubation time is indicated next to temperature. The growth curves of the wild-type and ΔtrmB strains in liquid cultures at 70°C (B), 80°C (C) and 70–80°C (D). The arrow in panel D indicates the shift point of temperature from 70°C to 80°C.

Figure 4.

Protein synthesis activities in the wild-type and ΔtrmB strain. The protein synthesis activities of the wild-type (left) and ΔtrmB (right) strains were compared by ³⁵S-Met incorporation at 70°C (A) and 70–80°C (B). ³⁵S-Met was added at the zero points and samples were taken out at 2, 5, 10, 15, 20 and 30 min. Total proteins were analyzed by 15% SDS–PAGE. The gels were stained with Coomassie brilliant blue (upper). ³⁵S-Met incorporation was monitored with a Fuji-photo film imaging analyzer (lower). Non RI means the sample before addition of ³⁵S-Met.

Figure 5.

The contents of modified nucleosides in class I tRNA from the ΔtrmB cells cultured at 70–80°C decrease. The modified nucleosides in class I tRNA from the wild-type (A) and the ΔtrmB (B) cells cultured at 70–80°C were compared. The content of each modified nucleosides was calculated as described in the ‘Materials and methods’ section, and is depicted in the figure.

Figure 6.

Methyl-transfer activities of tRNA methyltransferases for the RNAs from the wild-type and ΔtrmB strains. (A) Modified nucleotides are indicated in a tRNA cloverleaf structure together with the responsible modification enzymes (TrmB, TrmA, TrmD, TrmH and TrmI). Sources of the enzymes are indicated in parenthesis. (B) Purified tRNA methyltransferases (TrmB, TrmA, TrmD, TrmH and TrmI) were analyzed by 15% SDS–PAGE and the gel was stained with Coomassie brilliant blue. (C) ¹⁴C-methyl group acceptance activity of RNAs from the wild-type (white) and ΔtrmB (grey) strain. The ΔtrmB + m⁷G46 (black) annotation on the graph means the methyl acceptance activity of the RNA from the ΔtrmB strain methylated with TrmB. The methyl group acceptance activities at a 60 min period are shown. With the exception of the Gm18 modification by TrmH the graphs represent the apparent initial velocities. The methylation velocities except that for the Gm18 modification were relatively slow because the modified tRNA, which is abundant in the RNA, inhibits the modifications. (D) Methyl group incorporation catalyzed by TrmH was monitored in a time-dependent manner.

Figure 7.

Phe charging activities of purified tRNA^Phe. Phe charging activities at 70°C (A) and 80°C (B) were measured by ¹⁴C-Phe incorporation into the purified tRNA^Phe from the wild-type (filled circles) and ΔtrmB (open circles) strains. These graphs show one set of data from two independent experiments.

Figure 8.

Degradation of tRNA^Phe (UUC) and tRNA^Ile (AUC) in the ΔtrmB cells cultured at 70–80°C. (A) Small RNA fractions of the wild-type (left) and ΔtrmB (right) strains were time-dependently prepared from the cells cultured at 70–80°C. The zero periods mean the shift points of the culture temperature from 70°C to 80°C. 0.02 A₂₆₀ units of RNA samples were analyzed by 10% PAGE (7 M urea). The gels were stained with toluidine blue. (B) The RNA samples in panel A were analyzed by northern hybridization. Sequences of the DNA probes for tRNA detection are described in the ‘Materials and Methods’ section.

Figure 9.

Summary of this study. Effects of the trmB gene disruption are depicted. The lack of the m⁷G46 modification causes hypo-modification of other nucleotides in class I tRNA. The melting temperature of the tRNA decreases, and tRNA^Phe and tRNA^Ile are degraded. The degradation of tRNA depresses protein synthesis and the ΔtrmB strain exhibits a severe growth defect at high temperatures. These experimental results suggest the existence of a tRNA modification network, in which the m⁷G46 modification catalyzed by TrmB may act as one of the key factors.