Regulatory Factors for tRNA Modifications in Extreme- Thermophilic Bacterium Thermus thermophilus

Thermus thermophilus is an extreme-thermophilic bacterium that can grow at a wide range of temperatures (50–83°C). To enable T. thermophilus to grow at high temperatures, several biomolecules including tRNA and tRNA modification enzymes show extreme heat-resistance. Therefore, the modified nucleosides in tRNA from T. thermophilus have been studied mainly from the view point of tRNA stabilization at high temperatures. Such studies have shown that several modifications stabilize the structure of tRNA and are essential for survival of the organism at high temperatures. Together with tRNA modification enzymes, the modified nucleosides form a network that regulates the extent of different tRNA modifications at various temperatures. In this review, I describe this network, as well as the tRNA recognition mechanism of individual tRNA modification enzymes. Furthermore, I summarize the roles of other tRNA stabilization factors such as polyamines and metal ions.


INTRODUCTION
Thermus thermophilus is an extreme-thermophilic bacterium isolated from Mine Hot Spring in Japan that can grow at a wide range of temperatures (50-83 • C) (Oshima and Imahori, 1974). This bacterium can grow under aerobic conditions and possesses only about 2200 genes. Therefore, T. thermophilus strain HB8 was selected as a model organism in the Structural-Biological Whole Cell Project in Japan (Yokoyama et al., 2000). A method for preparing gene disruptant strains of T. thermophilus has been established (Hoseki et al., 1999;Hashimoto et al., 2001). Furthermore, both expression vectors for T. thermophilus proteins in Escherichia coli cells and gene disruption vectors are available from RIKEN Bio Resource Center 1 . Today, T. thermophilus is one of the most studied thermophiles.
Given that T. thermophilus grows at high temperatures, its biomolecules including tRNA and tRNA modification enzymes show extreme heat-resistance. As a result, modified nucleosides in tRNA from T. thermophilus have been studied from the viewpoint of stabilization of tRNA structure at high temperatures. In this review, I focus on the thermal adaptation system of tRNA modifications in T. thermophilus, which is regulated by many factors.  The m 5 s 2 U modification is a typical thermophile-specific modified nucleoside in tRNA . It was originally identified in RNase T 1 -digested RNA fragments derived from a tRNA mixture from T. thermophilus and sequence of the corresponding fragment strongly suggested that a portion of m 5 U at position 54 is replaced by m 5 s 2 U (Watanabe et al., 1974). Subsequently, it was found that this modified nucleoside was increased according to increasing in temperature of the cultures and melting temperature of tRNA was risen with increasing in the extent of m 5 s 2 U in tRNA (Watanabe et al., 1976). The presence of m 5 s 2 U54 was first confirmed in tRNA Met f 1 and tRNA Met f 2 , and then tRNA containing m 5 s 2 U54 was separated from tRNA containing m 5 U54 (Watanabe et al., 1983). The m 5 s 2 U54 modification has been identified in all T. thermophilus tRNA species sequenced so far [tRNA Ile 1 (Horie et al., 1985), tRNA Asp (Keith et al., 1993) and tRNA Phe (Grawunder et al., 1992;Tomikawa et al., 2010)]. hydrophobic interaction between the m 5 s 2 U54-A58 (or m 1 A58) and G53-C61 base pairs stabilizes the tertiary G18-ψ55 and G19-C56 base pairs between the T-and D-arms. Therefore, m 5 s 2 U54 contributes to stabilization of the L-shaped tRNA structure, and the melting temperature of tRNA is increased more than 3 • C by the presence of m 5 s 2 U54 (Watanabe et al., 1976; Mueller et al., 1998;Kambampati and Lauhon, 1999 Gm18 2 -O-methylguanosine TrmH Persson et al., 1997;Hori et al., 2002 D20 dihydrouridine DusA Bishop et al., 2002;Kusuba et al., 2015;Bou-Nader et al., 2018 i 6 A37 N 6 -isopentenyladenosine MiaA Caillet and Droogmans, 1988 ms 2 i 6 A37 2-methylthio-N 6 -isopentenyladenosine MiaA and MiaB Esberg et al., 1999;Pierrel et al., Watanabe et al., 1974;Shigi et al., 2006aShigi et al., , 2016Shigi, 2012 ψ55 pseudouridine TruB Nurse et al., 1995;Ishida et al., 2011 m 1 A58 N 1 -methyladenosine TrmI Droogmans et al., 2003;Takuma et al., 2015Davanloo et al., 1979. Furthermore, the melting temperature of a tRNA mixture is maintained above 85 • C by the 2thiomodification in m 5 s 2 U54 (Shigi et al., 2006b), enabling T. thermophilus to grow at temperatures of 50-83 • C.
Temperature-Dependent Regulation of m 5 s 2 U54 Content in tRNA Phe and Protein Synthesis Figure 3, which is based on a combination of previous experimental results, shows the percentage of different modified nucleosides (m 5 s 2 U54, Gm18, m 7 G46, and m 1 A58) in tRNA Phe , purified from T. thermophilus cells cultured at 50, 60, 70, and 80 • C (Tomikawa et al., 2010;Ishida et al., 2011;Yamagami et al., 2016). As shown in Figure 3, the extent of m 5 s 2 U54 in tRNA Phe increases with increasing culture temperatures. The balance between m 5 s 2 U54 and m 5 U54 regulates the rigidity (flexibility) of tRNA at a wide range of temperatures (50-83 • C). The presence of m 5 s 2 U54 in tRNA is required for efficient protein synthesis at high temperatures. A previous study measured the activity of Poly (U)-dependent poly-phenylalanine synthesis at several temperatures using various fractions (tRNA Phe , ribosome, and supernatant of 100,000 × g centrifugation fraction) prepared from T. thermophilus cells cultured at 50 and 80 • C (Yokoyama et al., 1987). Polyphenylalanine was effectively synthesized at 80 • C only when tRNA Phe from cells cultured at 80 • C was used. As shown in Figure 3, the proportion of m 5 s 2 U54 in tRNA Phe from cells cultured at 80 • C is higher than that in tRNA Phe from cells cultured at 50 • C. The melting temperature of a tRNA mixture from cells cultured at 70 • C is 79.7 • C in the presence of 50 mM Tris-HCl (pH7.5), 5 mM MgCl 2 and 100 mM NaCl (Tomikawa et al., 2010); thus, the structure of tRNA Phe from cells cultured at 50 • C seems to be looser than that from cells cultured at 80 • C. Indeed, the gene disruption strain of ttuA encoding a component of sulfur-transfer complex for the m 5 s 2 U54 formation, results in a growth defect of the T. thermophilus strain at 80 • C (Shigi et al., 2006a). Thus, the m 5 s 2 U54 modification is essential for effective protein synthesis in T. thermophilus at high temperatures. FIGURE 3 | Extents of m 7 G46, m 1 A58, Gm18, and m 5 s 2 U54 modifications in tRNA Phe from T. thermophilus. The extent of m 7 G46, m 1 A58, and Gm18 modifications was measured by a methylation assay with TrmB, TrmI, and TrmH, respectively. The extent of m 5 s 2 U54 modification was estimated from the peak areas of m 5 U54 and m 5 s 2 U54 on HPLC analysis. This figure is prepared from Figure 3 in a chapter "Regulation of Protein Synthesis via the Network Between Modified Nucleotides in tRNA and tRNA Modification Enzymes in T. thermophilus, a Thermophilic Eubacterium" of a book "Modified Nucleic Acids in Biology and Medicine," Springer Nature 2016 with permission (4517441319562) from the publisher. m 5 s 2 U54 in tRNA From Other Thermophiles In addition to T. thermophilus, m 5 s 2 U has been identified among the modified nucleosides in unfractionated tRNA from Thermotoga maritima (Edmonds et al., 1991) and at position 54 in tRNA Cys from Aquifex aeolicus (Awai et al., 2009). Furthermore, the m 5 s 2 U nucleoside has been identified among the modified nucleosides in unfractionated tRNA from some hyper-thermophilic archaea such as Thermococcus species (Edmonds et al., 1991) and Pyrococcus furiosus (Kowalak et al., 1994). Therefore, it is considered that these hyper-thermophiles also possess the m 5 s 2 U54 modification in tRNA.

Biosynthetic Pathway of m 5 s 2 U54 in Eubacteria and Archaea
Biosynthesis of m 5 s 2 U54 is accomplished in two steps, methylation of the C5 atom and the 2-thiolation. Although these steps occur independently at U54 in tRNA (Shigi et al., 2006a;Yamagami et al., 2016), the m 5 U54 modification in tRNA is almost fully formed in living cells even when T. thermophilus is cultured under nutrient-poor conditions . To date, therefore, an s 2 U54 modification has not been observed in tRNA from the T. thermophilus wild-type strain.
The 2-thiolation in m 5 s 2 U54 of T. thermophilus is conferred by multiple proteins, namely TtuA, TtuB, TtuC, TtuD and IscS (or SufS) (Shigi et al., 2006a(Shigi et al., , 2008Shigi, 2012;Chen et al., 2017). In addition, the mechanism of the sulfurtransfer reaction carried out by TtuA from T. maritima has been recently proposed based on crystal structures of the enzyme (Arragain et al., 2017). The protein factors involved in 2-thiolation of m 5 s 2 U54 in archaea have not been confirmed experimentally.

tRNA MODIFICATION ENZYMES RECOGNIZES THE LOCAL STRUCTURE(S) IN tRNA
At the beginning of this century, the mechanisms of regulating the extent of modified nucleosides in tRNA from T. thermophilus were unknown. The transcriptional and/or translational regulations of amounts of tRNA modification enzyme(s) were assumed at the start of our studies, however, we noticed that this regulation might be explainable by the substrate tRNA recognition mechanisms of the tRNA modification enzymes. In general, tRNA modification enzymes recognize the local structure in tRNA. Figure 4 shows the minimum substrate or positive determinants for different tRNA modification enzymes, which I describe in more detail below.

m 5 U54 Formation by TrmFO
TrmFO can methylate a micro-helix RNA, which mimics the T-arm structure (Yamagami et al., 2012; Figure 4A). The positive determinants for TrmFO are the stem-loop structure, G53-C61 base pair, and U54U55C56 sequence. Therefore, the substrate RNA recognition mechanism of TrmFO is very simple. This simple structure is also observed in the anticodon-loop of tRNA Pro from T. thermophilus; however, A38 in the anticodonloop prevents incorrect methylation by TrmFO (Yamagami et al., 2012). In some cases, therefore, there are negative determinants for tRNA modification enzymes.

s 2 U54 Formation by TtuA
In the case of TtuA (Figure 4B), only the modification patterns of tRNA Asp mutants expressed in T. thermophilus cells have been analyzed (Shigi et al., 2002); therefore, it is unknown whether TtuA can act on a micro-helix RNA. Nevertheless, it is clear that the positive determinants for the sulfur-transfer reaction of TtuA are also very simple. More recently, it was shown that the presence of m 1 A58 accelerates the velocity of sulfur-transfer (Shigi et al., 2006b). Thus, the sulfur-transfer reaction carried out by TtuA is regulated by another modification (m 1 A58).

m 1 A58 Formation by TrmI
The tRNA m 1 A58 methyltransferase TrmI can methylate a micro-helix RNA very slowly (Takuma et al., 2015; Figure 4C). The presence of an aminoacyl-stem or variable region accelerates the rate of methylation of truncated tRNA by TrmI. Furthermore, the presence of an m 7 G46 modification also accelerates the rate of methylation by TrmI (Tomikawa et al., 2010). Thus, the extent of m 1 A58 modification in tRNA is also controlled by the presence of another modification (m 7 G46).
Among T. thermophilus tRNAs, tRNA Thr GGU exceptionally possesses C60 instead of U60, which is one of the positive determinants for TrmI. The proportion of m 1 A58 and m 5 s 2 U54 in tRNA Thr GGU  is lower than that in tRNA Phe (Takuma et al., 2015), indicating that the extent of m 1 A58 modification has an effect on the extent of m 5 s 2 U54 modification, which is consistent with the observation of Shigi et al. (2006b). Thus, the degree of m 1 A58 modification in tRNAs differs according to the sequence of each tRNA. The sequence of tRNA Thr GGU seems to be disadvantageous for survival of T. thermophilus at high temperatures, however, the physiological reason for the presence of C60 in tRNA Thr GGU is unknown.
ψ55 Formation by TruB Escherichia coli TruB can modify a micro-helix RNA, which mimics the T-arm structure (Gu et al., 1998). U54, U55, pyrimidine56 and A58 in the T-loop are important for the full activity of E. coli TruB ( Figure 4D; Gu et al., 1998). The crystal structure of a complex of TruB and micro-helix RNA showed that other nucleotides in the T-loop contact with TruB (Hoang and Ferré-D'Amaré, 2001;Pan et al., 2003). The ribosephosphate backbone in the T-arm structure, which is formed by the U54-A58 reverse Hoogsteen base pair, is important for the reaction by E. coli TruB. Although the substrate tRNA recognition mechanism of T. thermophilus TruB has not been confirmed experimentally, the high conservation of amino acid sequences between E. coli and T. thermophilus TruB proteins (Ishida et al., 2011) strongly suggests that these enzymes possess a common mechanism of tRNA recognition. Formation of ψ55 in T. thermophilus tRNA Phe transcript by TruB is very rapid at 55 • C (Ishida et al., 2011), suggesting that T. thermophilus TruB does not require other modifications in tRNA in order to function.

m 7 G46 Formation by TrmB
Aquifex aeolicus TrmB can methylate a truncated tRNA ( Figure 4E): its methylation speed for truncated RNA is comparable to that for the full-length tRNA transcript (Okamoto et al., 2004). In the truncated tRNA, the T-arm-like structure and five nucleosides corresponding to variable region are essential for methylation by TrmB. Thermophilic TrmB (A. aeolicus and T. thermophilus TrmB) share considerable amino acid sequence homology and exceptionally possess a long C-terminal region (Okamoto et al., 2004), which is involved in binding to AdoMet . Therefore, the substrate tRNA recognition mechanism of thermophilic TrmB may be different from that of mesophilic TrmB (De Bie et al., 2003;Purta et al., 2005;Zegers et al., 2006;Zhou et al., 2009;Tomikawa, 2018). Figure 4F shows the minimum substrate RNA of A. aeolicus TrmH (Hori et al., 2003). There are some differences in substrate tRNA recognition mechanism between A. aeolicus TrmH and T. thermophilus TrmH. For example, A. aeolicus TrmH cannot methylate tRNAs with A17 (Hori et al., 2003), but T. thermophilus TrmH methylates tRNA Ser , which contains A17 (Hori et al., 1998). Furthermore, the size and sequence of the D-loop have effects on methylation by A. aeolicus TrmH (Hori et al., 2003), however, T. thermophilus TrmH methylates mutant tRNAs irrespective of these D-loop features (Ochi et al., 2010). In short, T. thermophilus TrmH has been found to methylate all tRNAs tested so far (Hori et al., 1998). T. thermophilus TrmH can methylate a 5'-half fragment of tRNA (Matsumoto et al., 1990) but not a micro-helix that mimics the D-arm (Hirao et al., 1989). Therefore, T. thermophilus TrmH may recognize the bulge structure like A. aeolicus TrmH. This idea is consistent with observations that the cross-linking or chemical modification of s 4 U8 in substrate tRNA causes a decrease in methylation speed by TrmH . Full activity of TrmH requires the L-shaped structure formed by conserved nucleosides in tRNA (Hori et al., 1998). Furthermore, the speed of methylation by T. thermophilus TrmH for yeast tRNA Phe transcript is slower than that for native yeast tRNA Phe at 65 • C, showing that other modified nucleosides have a positive effect on the methylation by TrmH (Hori et al., 1998). Indeed, the presence of m 7 G46 in tRNA Phe transcript accelerates methylation speed by TrmH (Tomikawa et al., 2010).

s 4 U8 Formation by ThiI
Escherichia coli and T. maritima ThiI (tRNA 4-thiouridine synthetase) can modify several truncated tRNAs that mimic the aminoacyl-stem and T-arm ( Figure 4G; Lauhon et al., 2004;Neumann et al., 2014). Although the substrate tRNA recognition mechanism of T. thermophilus ThiI has not been investigated, it is likely that T. thermophilus ThiI also recognizes the local structure in tRNA. The THUMP domain in ThiI recognizes the CCA terminus of substrate RNA (Neumann et al., 2014). This feature has been identified in another tRNA modification enzyme, archaeal Trm11, which also possesses a THUMP domain (Hirata et al., 2016). Furthermore, given that TrmN and archaeal Trm14 have a THUMP domain (Menezes et al., 2011;Fislage et al., 2012;Roovers et al., 2012), these enzymes may recognize the CCA terminus in tRNA.

D20 and D20a Formations by DusA
In T. thermophilus, single Dus family protein, DusA synthesizes all D modifications (D20 and D20a) in tRNA (Kusuba et al., 2015); in E. coli, by contrast, three Dus family proteins share the modification sites in tRNA (Bishop et al., 2002;Bou-Nader et al., 2018). Among the T. thermophilus tRNA modification enzymes that act on the three-dimensional core in tRNA, DusA exceptionally recognizes the interaction between the T-arm and D-arm (Yu et al., 2011). For the reaction of DusA at high temperatures, therefore, stabilization of the L-shaped tRNA structure by other modified nucleosides is essential (Kusuba et al., 2015). Thus, D20 and D20a seem to be relatively late modifications in T. thermophilus tRNA.

INITIAL BINDING AND INDUCED-FIT STEPS IN COMPLEX FORMATION BETWEEN tRNA MODIFICATION ENZYMES AND SUBSTRATE tRNA
As shown in Figure 4, many tRNA modification enzymes recognize the local structure in tRNA, while the interaction between the T-arm and D-arm in tRNA is not required for their activity. Indeed, in the case of TrmI, the methylation speed is faster for a mutant tRNA transcript, in which the interaction between the T-arm and D-arm is disrupted, than for the wild-type tRNA transcript (Takuma et al., 2015). In many cases, the target modification site is embedded in the L-shaped tRNA structure. For example, G18 and U55 form a tertiary base pair and the uracil base in U55 is not localized at the surface of tRNA. Therefore, disruption of L-shaped tRNA structure is necessary for the reaction of TruB. Furthermore, to fit into the catalytic pocket of TruB, the uracil base must be flipped. These observations suggest that the reaction of tRNA modification enzyme comprises at least two steps, initial binding to the L-shaped tRNA, followed by a structural change (inducedfit) process in which the L-shaped tRNA structure is disrupted.
These steps have been monitored for complex formation between T. thermophilus TrmH and tRNA Phe transcript by using a stopped-flow fluorescence measurement system (Figure 5; Ochi et al., 2010Ochi et al., , 2013. T. thermophilus TrmH is a member of the SpoU-TrmD (so-called SPOUT) methyltransferase superfamily (Anantharaman et al., 2002;Nureki et al., 2004;Hori, 2017), and site-directed mutagenesis studies (Nureki et al., 2004;Watanabe et al., 2005) suggest that arginine at position 41 (Arg41) in TrmH acts as the catalytic center. As shown in Figure 5A, TrmH is a dimeric enzyme with three tryptophan residues (Trp73, Trp126, and Trp191) in each subunit. The fluorescence intensity at 320 nm derived from these tryptophan residues was measured during complex formation between TrmH and tRNA. Figure 5B shows the one result obtained when 7.70 µM TrmH-AdoMet complex and 7.70 µmM tRNA Phe transcript were mixed by the stopped-flow system at 25 • C (Ochi et al., 2010). A very fast decrease in fluorescence was observed in the initial 10 ms, followed by relatively slow increase in fluorescence from 10 to 50 ms. In general, a decrease of tryptophan fluorescence intensity suggests an increase in accessibility of the residue to solvent water. The obtained data could be fitted to an equation, which showed that the reaction was bi-molecular binding reaction. In the case of TrmH, therefore, the initial decrease of fluorescence suggests that a tryptophan residue(s) is moved to the solvent during the initial binding process. Subsequently, this residue was confirmed as Trp126 by measurements on TrmH mutant proteins (Ochi et al., 2013). The slow increase in fluorescence from 10 to 50 ms reflects the structural change (induced-fit) process; detailed measurements on several concentrations of TrmH and tRNA indicate that the slow increase in fluorescence is fitted to a combination of unimolecular (first-order) reactions. The methylation was not completed within 50 ms, showing that the slow increase in fluorescence was not caused by dissociation of tRNA from the enzyme after the reaction. In the structural change process, Trp126 is moved to a hydrophilic environment. During the induced fit process, disruption of L-shaped tRNA structure, recognition of 6-oxygen in G18, and introduction of ribose into the catalytic pocket are occurred (Ochi et al., 2010).
The folding of tRNA and rigidity (flexibility) of the local structure in tRNA affect the speed of the initial binding and induced-fit processes. Thus, other modifications in tRNA, temperature, and RNA stabilization factors such as Mg 2+ ions and polyamines are all likely to influence on the initial binding and induced-fit processes.

A NETWORK BETWEEN MODIFIED NUCLEOSIDES IN tRNA AND tRNA MODIFICATION ENZYMES CONTROLS THE FLEXIBILITY (RIGIDITY) OF tRNA IN T. thermophilus AT A WIDE RANGE OF TEMPERATURES
A method for preparing gene disruptant strain of T. thermophilus was developed at the beginning of this century (Hoseki et al., 1999;Hashimoto et al., 2001). This gene-disruption system, coupled with biochemical studies, has been used to elucidate the regulatory network between modified nucleosides in tRNA and tRNA modification enzymes in T. thermophilus cells. Figure 6 summarizes the network between modified nucleosides in tRNA and tRNA modification enzymes. Although each tRNA modification enzymes can act on unmodified tRNA transcript (or truncated tRNA transcript as shown in Figure 4), the presence of modified nucleosides often accelerates (or slows down) the speed of modification by other tRNA modification enzymes depending on the environmental temperatures.
T. thermophilus lives in hot springs. The temperature of hot spring water can change for several reasons including an influx of river water, snowfall, and eruption of hot water. Therefore, the ability of protein synthesis to adapt to temperature changes via the flexibility (rigidity) of tRNA is very important for survival of T. thermophilus. One of advantages of the network is that it does not require protein synthesis. As a result, it can respond rapidly. Furthermore, the network may be a survival strategy of eubacteria, which have a limited genome size.

Network at High Temperatures (>75 • C)
At high temperatures (>75 • C), m 7 G46 modification by TrmB is one of the key modifications in the network (Figure 6A). It has been shown that the trmB gene deletion strain does not grow at 80 • C and has several hypo-modifications in tRNA (Tomikawa et al., 2010). When the culture temperature is shifted from 70 to 80 • C, tRNA Phe and tRNA Lys are degraded and protein synthesis is impaired in this strain. Particular, heat shock proteins are not synthesized efficiently in the trmB gene deletion strain. Thus, the m 7 G46 modification is essential for survival of T. thermophilus at high temperatures.
The positive effects of m 7 G46 on TrmH, TrmD, and TrmI activity have been confirmed by in vitro experiments. Because the m 1 G37 modification conferred by TrmD is not present in T. thermophilus tRNA Phe , yeast tRNA Phe transcript was used in these experiments (Tomikawa et al., 2010). TrmH can methylate a 5'-half fragment of tRNA (Matsumoto et al., 1990), but its full activity requires the three-dimensional core structure of tRNA (Hori et al., 1998). m 7 G46 forms a tertiary base pair with the C13-G22 base pair in the D-arm; thus, this tertiary base pair seems to have a positive effect on TrmH activity. TrmD can act on a truncated tRNA (Redlak et al., 1997) and micro-helix RNA (Takeda et al., 2006), but foot-printing analyses have shown that the D-arm and variable region are protected in addition to the anticodon-arm (Gabryszuk and Holmes, 1997). Furthermore, the crystal structure of the TrmD-tRNA complex revealed that the C-terminal domain of TrmD makes contacts with the D-arm in tRNA (Ito et al., 2015). Therefore, the positive effect of m 7 G46 on TrmD activity can be explained by stabilization of the D-arm via the formation of an m 7 G46-C13-G22 tertiary base pair.
In the case of TrmI, the presence of aminoacyl-stem or variable region increases the methyl-group acceptance activity of a truncated tRNA transcript (Takuma et al., 2015). Although there is no docking model of TrmI with tRNA, positivelycharged grooves, which are present on the surface of TrmI (Barraud et al., 2008), may capture the aminoacyl-stem and variable region in tRNA.
The positive effect of m 5 U54 on TrmI activity was also confirmed in vitro (Yamagami et al., 2012). However, the tRNA fraction from a trmFO gene deletion strain cultured at 70 • C contained the same amount of m 1 A nucleoside as that from the wild-type strain (Yamagami et al., 2016). Therefore, there seems to be a sufficient amount of TrmI to maintain the extent of m 1 A58 in tRNA in the T. thermophilus trmFO gene deletion strain. The . The m 7 G46 modification (highlighted in red) is a key factor in this network. Its presence accelerates the speed of other tRNA modification enzymes such as TrmH, TrmD, and TrmI. In addition, the presence of m 5 U54 also increases the methylation speed of TrmI. The increase in m 1 A58 due to accelerated TrmI activity further increases the speed of sulfur-transfer by TtuA and that of related proteins, and results in an increased percentage of m 5 s 2 U54. The introduced modifications coordinately stabilize the L-shaped tRNA structure. (B) Network at low temperatures (<55 • C). In this network, the ψ55 modification stabilizes the local structure in tRNA and slows down the speed of tRNA modification enzymes. The m 5 U54 modification plays a role in maintaining the balance of modifications at the elbow region in tRNA. This figure is prepared from Figure 4 in a chapter "Regulation of Protein Synthesis via the Network Between Modified Nucleotides in tRNA and tRNA Modification Enzymes in T. thermophilus, a Thermophilic Eubacterium" of a book "Modified Nucleic Acids in Biology and Medicine" Springer Nature 2016 with permission (4517441319562) from the publisher. positive effect of m 5 U54 on the TrmI activity can be explained by stabilizing effect of the m 5 U54-A58 reverse Hoogsteen base pair.
As described in the Section "tRNA Modification Enzymes Recognizes the Local Structure(s) in tRNA", the m 1 A58 modification conferred by TrmI is a positive determinant for the sulfur-transfer system (TtuA and related proteins) (Shigi et al., 2006b). Therefore, TrmI is essential for survival of T. thermophilus at high temperatures (Droogmans et al., 2003). Furthermore, the m 5 s 2 U54 modification is essential for stabilizing the tRNA structure, as described in the Section "The m 5 s 2 U54 Modification in T. thermophilus tRNA is Essential for Protein Synthesis at High Temperatures." As a result, the sulfurtransfer system for s 2 U54 formation is also essential for the survival of T. thermophilus at high temperatures (Shigi et al., 2006a). In contrast, TrmFO is not essential and the trmFO gene deletion strain can grow at 80 • C (Yamagami et al., 2016). Collectively, these modified nucleosides in tRNA coordinately stabilize the tRNA structure at high temperatures.
Given that the formation of D20 by DusA requires the interaction between D-arm and T-arm (Yu et al., 2011), stabilization of the L-shaped tRNA structure is essential for D20 formation at high temperatures (Kusuba et al., 2015). Therefore, several modifications, including m 5 s 2 U54, m 1 A58, Gm18 and ψ55, seem to be required for sufficient activity of DusA at high temperatures.
Among the modified nucleosides in T. thermophilus tRNA Phe , m 2 G6 by TrmN, s 4 U8 by ThiI, i 6 A37 and ms 2 i 6 A37 by MiaA and MiaB, and ψ39 by TruA have not been investigated as yet. However, it is possible that some of them may affect the stability of tRNA in T. thermophilus at high temperatures. For example, it has been recently reported that the melting temperature of tRNA from E. coli thiI gene disruptant strain is lower than that from the wild-type strain (Nomura et al., 2016). Therefore, s 4 U8 may contribute to stabilization of the structure of tRNA. In the case of anticodon-loop modifications (ψ38, i 6 A37, and ms 2 i 6 A37), their deletion may severely impair protein synthesis because they are important for the structure of anticodon-loop and function directly in protein synthesis (Spenkuch et al., 2014;Grosjean and Westhof, 2016;Schweizer et al., 2017). Furthermore, i 6 A37 is required for the 2'-Omethylation conferred by TrmL at position 34 in E. coli tRNA Leu (Benítez-Páez et al., 2010;Zhou et al., 2015). In the case of T. thermophilus tRNA Leu , therefore, TrmL is probably included in the network.

Network at Low Temperatures (<55 • C)
At low temperatures (<55 • C), the ψ55 modification conferred by TruB works as a key factor in the network (Figure 6B). In the truB gene deletion strain, excess amounts of Gm18, m 1 A58 and m 5 s 2 U54 are introduced into tRNAs at low temperatures and the melting temperature of tRNA mixture is increased by more than 8 • C (Ishida et al., 2011). This excess rigidity of tRNA results in a disorder of protein synthesis, and cold shock proteins are not synthesized efficiently in the truB gene deletion strain. Therefore, ψ55 in tRNA is required for survival of T. thermophilus at low temperatures.
The m 5 U54 modification aids ψ55 in maintaining the balance of other modifications in tRNA (Yamagami et al., 2016). The ψ55 modification stabilizes the structure of elbow region in tRNA and slows down the formation speed of other modifications around ψ55 (Gm18, m 1 A58 and m 5 s 2 U54) (Ishida et al., 2011). The positive effect of m 5 U54 on m 1 A58 modification was confirmed both by the in vivo methylation profile and by in vitro experiments (Yamagami et al., 2016) and is probably due to the stabilization of the m 5 U54-A58 reverse Hoogsteen base pair.
WHAT STABILIZES THE STRUCTURE OF UNMODIFIED PRECURSOR tRNA IN T. thermophilus AT 80 • C?
Unmodified tRNA transcript cannot maintain its L-shaped tRNA structure at high temperatures. Primary transcript (precursor tRNA), which is synthesized by RNA polymerase, is unmodified. Therefore, even though several tRNA modification enzymes from T. thermophilus can act on unmodified tRNA transcript, their activities cannot be measured at 80 • C due to the disrupted structure of substrate RNA. For example, T. thermophilus TrmH methylates tRNA effectively only at temperatures below the melting temperature (Matsumoto et al., 1987). These observations raise an important question, what stabilizes the structure of unmodified precursor tRNA in T. thermophilus at 80 • C? If there were no stabilization factors in living cells, tRNA modification enzymes from T. thermophilus would not be able to act on precursor tRNA at 80 • C.
Because polyamines have positive charges and hydrophilic regions, they have the potential to interact with nucleic acids and phospholipids. Indeed, there have been several studies on the interaction between polyamines and tRNA. For example, addition of polyamines shifted the melting temperature of native tRNA Phe from Saccharomyces cerevisiae to higher temperatures in accordance with polyamine length . The crystal structure of the complex of yeast tRNA Phe and spermine revealed that two spermine molecules bind to two sites in one tRNA Phe molecule (Quigley et al., 1978). Furthermore, FT-IR analysis of tRNA in the presence of putrescine, spermidine, and spermine showed that similar to spermine, putrescine and spermidine bind to the connection region between the D-arm and anticodon-stem (Ouameur et al., 2010). Moreover, a 13 C-NMR study reported that 14 spermidinebinding sites were present in one tRNA molecule and that three spermidine molecules stably bound to tRNA in the presence of Mg 2+ (Frydman et al., 1990). It has also been reported that a branched polyamine (Figure 7A), tetrakis(3aminopropyl)ammonium (Taa), slightly stimulates the activity of archaeal Trm1 and TrmI (Hayrapetyan et al., 2009), which are archaeal tRNA methyltransferases for the formation of m 2 2 G26 (or m 2 G26) (Constantinesco et al., 1999) and m 1 A57 and m 1 A58 (Roovers et al., 2004), respectively.

TrmH Methylates Unmodified tRNA Transcript at 80 • C Only in the Presence of Long or Branched Polyamine
The effect of polyamine on the methyl-transfer speed of TrmH for yeast tRNA Phe transcript has been measured at various temperatures ( Figure 7B; Hori et al., 2016). All polyamines were found to increase the speed of methylation by TrmH at appropriate temperatures, although the positive effect of putrescine was relatively weak and observed only at low temperatures. As the length of polyamine increased, the optimum temperature for methyl-transfer shifted to higher temperatures, however, standard polyamines did not work at 80 • C. By contrast, very weak but clear methyl-transfer activity of TrmH for unmodified tRNA Phe was observed at 80 • C in the presence of optimum concentration (1.5 mM) of caldohexamine, a long polyamine, (red arrow in Figure 7B). Addition of branched polyamine Taa had a stronger positive effect on TrmH activity at 80 • C (red arrow in Figure 7B). Thus, long and branched polyamines can support the methylation by TrmH at 80 • C.
If initial modifications are introduced into unmodified precursor tRNA, they stabilize the local structure in tRNA enabling the tRNA modification enzymes to function on the precursor tRNA. Introduction of initial modifications into tRNA probably occurs in the presence of polyamines. Among polyamines, long and branched polyamines are effective at very high temperatures.
Because TrmB, which confers the m 7 G46 modification, is a key enzyme in the network at high temperatures, the effects of polyamines on TrmB activity should be clarified. At present, however, this is not possible due to a technical problem: T. thermophilus TrmB, which is expressed in E. coli cells, is partially degraded in these cells and purification of intact TrmB is difficult.

Long and Branched Polyamines Are Required for Maintenance of 70S Ribosome and Several tRNAs
The biosynthetic pathway from arginine to spermidine in T. thermophilus is different from that in eukaryotes, archaea, and other bacteria because the intermediate in T. thermophilus is N 1 -aminopropylagmatine (Ohnuma et al., , 2011. S-Adenosyl-L-methionine decarboxylase-like protein 1 (SpeD1) is required for the biosynthesis of N 1 -aminopropylagmatine from arginine, and aminopropylagmatine ureohydrolase (SpeB) catalyzes the conversion of N 1 -aminopropylagmatine to spermidine. Because long and branched polyamines are synthesized from spermidine, the T. thermophilus speD1 or speB gene deletion strain cannot produce long and branched polyamines (Nakashima et al., 2017) and cannot grow at high temperatures (>75 • C) unless polyamines are added to the medium. When the speD1 and speB deletion strains were cultured at 70 • C in minimal medium until mid-log phase and then the culture temperature was shifted to 80 • C, they could survive for 10 h. Although abnormal modifications in tRNA were expected, at least the m 5 U54, m 7 G46, Gm18 and m 1 A58 modifications were present as normal in the tRNA mixture from these strains. Given that the transcription of tRNA and the introduction of major modifications into tRNA are expected to occur mainly before the mid-log phase, it seems that these modification can be introduced into tRNA at 70 • C without the presence of long and branched polyamines: the expression patterns of mRNAs in the wild-type strain can be obtained from the database (NCBI/GEO 2 ) (Shinkai et al., 2007). After the temperature shift, tRNA His , tRNA Tyr and 70S ribosome were gradually degraded in the speD1 and speB deletion strains and protein synthesis was severely impaired (Nakashima et al., 2017). Thus, long and branched polyamines are required to maintain several tRNAs at high temperatures in T. thermophilus in addition to regulating the extent of modified nucleosides in tRNA. 2 https://www.ncbi.nlm.nih.gov/gds/ Other Regulatory Factors for tRNA Stability RNA binding proteins, Mg 2+ ions and K + ions can stabilize tRNA structure.
In A. aeolicus, a hyper-thermophilic bacterium, tRNAbinding protein 111 (Trbp111) stabilizes the three-dimensional core of tRNA (Morales et al., 1999;Swairjo et al., 2000). However, Trbp111 is specific to A. aeolicus and not found in T. thermophilus. Archease is also an RNA-binding protein that changes the specificity of archaeal Trm4 (Auxilien et al., 2007) and is required for tRNA splicing (Desai et al., 2014;Popow et al., 2014). Although neither trm4 nor a tRNA gene with an intron-coding region is not encoded in the T. thermophilus genome, an archease-like protein gene (TTHA1745) exists. Therefore, it is possible that an archeaselike protein stabilizes tRNA structure at high temperatures in T. thermophilus cells.
Mg 2+ ions are required for folding of tRNA (Lorenz et al., 2017) and are important in considering the structural effects of several modifications in tRNA (Yue et al., 1994;Agris, 1996;Nobles et al., 2002). However, the precise concentrations of Mg 2+ ions in T. thermophilus cells are unknown. K + ions are also important for correct folding of tRNA. The concentrations of K + ions in the cells of several thermophilic archaea are reported to be extremely high (>700 mM) (Hensel and Konig, 1988). However, the intracellular concentrations of K + ions of T. thermophilus have not been reported. The concentrations of Mg 2+ and K + ions may change depending on the growth environment.

PERSPECTIVE
2018 marked the 50th anniversary year of the first isolation of T. thermophilus. In these past 50 years, T. thermophilus has been studied as a model organism that can adapt to extremely high temperatures. In this regard, the modifications in tRNA have been studied mainly from the viewpoint of the stabilization of tRNA structure at high temperatures. In particular, the role of m 5 s 2 U54 in tRNA has been clarified, the genes responsible for almost all tRNA modifications in T. thermophilus have been annotated and the regulatory network between modified nucleosides and tRNA modification enzymes has been identified. Furthermore, numerous tRNA modification enzymes from T. thermophilus have been used in structural studies (reviewed in Hori et al., 2018).
Nevertheless, several enigmas remain, even today. For example, the functions of tRNA modifications in the anticodonloop at high temperatures have not been studied in detail. Studies on anticodon-loop modifications are difficult because in vitro protein synthesis of T. thermophilus does not work effectively at high temperatures (>75 • C). Although there have been many attempts to synthesize proteins at high temperatures using a T. thermophilus cell-free translation system (Ohno-Iwashita et al., 1975;Uzawa et al., 1993a,b;Zhou et al., 2012), it remains difficult to monitor protein synthesis at 80 • C. As a result, our knowledge about the effects of tRNA modifications on codonanticodon interactions, on maintenance of reading frame and on dynamics of tRNA on ribosome at high temperatures is limited. Furthermore, recent studies on mesophiles reported that tRNA modifications occur in response to environmental stresses or function as stress resistance factors (Nawrot et al., 2011;Preston et al., 2013;Endres et al., 2015;Jaroensuk et al., 2016;Campos Guillen et al., 2017). As yet, however, there are no studies from this viewpoint for T. thermophilus. To understand the roles of tRNA modifications in totality, further studies will be required.

AUTHOR CONTRIBUTIONS
HH determined the concept of this review and prepared the manuscript.

FUNDING
This work was supported by a Grant-in-Aid for Scientific Research (16H04763 to HH) from the Japan Society for the Promotion of Science (JSPS).