International Journal of
Molecular Sciences
Review
7-Methylguanosine Modifications in
Transfer RNA (tRNA)
Chie Tomikawa
Department of Materials Science and Biotechnology, Graduate School of Science and Engineering,
Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan; tomikawa.chie.mm@ehime-u.ac.jp;
Tel./Fax: +81-89-927-9947
Received: 13 November 2018; Accepted: 13 December 2018; Published: 17 December 2018
Abstract:
More than 90 different modified nucleosides have been identified in tRNA. Among the
tRNA modifications, the 7-methylguanosine (m
7
G) modification is found widely in eubacteria,
eukaryotes, and a few archaea. In most cases, the m
7
G modification occurs at position 46 in the
variable region and is a product of tRNA (m
7
G46) methyltransferase. The m
7
G46 modification forms
a tertiary base pair with C13-G22, and stabilizes the tRNA structure. A reaction mechanism for
eubacterial tRNA m
7
G methyltransferase has been proposed based on the results of biochemical,
bioinformatic, and structural studies. However, an experimentally determined mechanism of
methyl-transfer remains to be ascertained. The physiological functions of m
7
G46 in tRNA have
started to be determined over the past decade. For example, tRNA m
7
G46 or tRNA (m
7
G46)
methyltransferase controls the amount of other tRNA modifications in thermophilic bacteria,
contributes to the pathogenic infectivity, and is also associated with several diseases. In this review,
information of tRNA m
7
G modifications and tRNA m
7
G methyltransferases is summarized and the
differences in reaction mechanism between tRNA m
7
G methyltransferase and rRNA or mRNA m
7
G
methylation enzyme are discussed.
Keywords: RNA modification; tRNA methyltransferase; tRNA modification; methylase
1. Introduction
Transfer RNA, which is one of the classical non-coding RNAs, functions as an adaptor molecule
supplying amino acids to ribosomes according to the codon of the mRNA. It is important that
tRNA forms a precise L-shape structure for its full function [
1
] and this requires tRNA modification.
More than 100 different modified nucleosides have been reported to date and found throughout
the different families of RNA molecules [
2
,
3
]. tRNA in particular is the most heavily modified [
3
,
4
].
The modified nucleosides include thiolation, deamination, isomerization conversion of uridine to
pseudouridine, or the combination of several modifications. Of the modification, methylation is the
most abundant. This modification encompasses 1-methyladenosine (m
1
A), 5-methyluridine (m
5
U),
5-methylcytidine (m
5
C), 1-, 2-, or 7- position methylation of G (m
1
G, m
2
G, m
7
G), 2’-O-methylation
of ribonucleoside (Nm), and others [
3
,
5
,
6
]. The most widely prevalent tRNA methylation is
S-adenosyl-L-methionine (AdoMet)-dependent methylation by AdoMet dependent methyltransferases.
7-methylguanosine (m
7
G) is one of the most conserved modified nucleosides and is common in
eubacteria, eukaryotes [
4
], and a few archaea [
7
]. Even in psychrophiles which have low levels of
modified nucleoside content, m
7
G has been found in addition to dihydrouridine (D), pseudouridine
(
Ψ
), and m
5
U [
8
]. Additionally, m
7
G is present in introns containing pre-tRNA together with
N
2
,N
2
-dimethylguanine (m
2
2
G),
Ψ
and m
1
A [
9
]. For this reason, it is thought that m
7
G is generated
immediately after the transcription. m
7
G is most frequently located at position 46 in the tRNA
variable region, and forms a tertiary base pair with C13-G22 in the three-dimensional core of tRNA
Int. J. Mol. Sci. 2018, 19, 4080; doi:10.3390/ijms19124080 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2018, 19, 4080 2 of 14
(The nucleotide positions in tRNA are numbered, according to the reference [
4
].) [
10
–
12
]. m
7
G has
no (net) charge under physiological conditions, but is positively charged in position 46 in tRNA via
hydrogen bonding to bases G22 and C13 [
13
–
15
] (Figure 1). Thus, 7-methylation of m
7
G produces
a site-specific electrostatic charge within the tRNA structure [
15
]. The tertiary base pair of the
m
7
G46-C13-G22 is considered to contribute to stabilization of the tRNA three-dimensional core.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 14
(net) charge under physiological conditions, but is positively charged in position 46 in tRNA via
hydrogen bonding to bases G22 and C13 [13–15] (Figure 1). Thus, 7-methylation of m
7
G produces a
site-specific electrostatic charge within the tRNA structure [15]. The tertiary base pair of the m
7
G46-
C13-G22 is considered to contribute to stabilization of the tRNA three-dimensional core.
Figure 1. tRNA m
7
G46 methyltransferase methylates the N7-atom of guanine at position 46 in tRNA
and forms m
7
G46. (A) The secondary structure of tRNA is presented in cloverleaf form. Conserved
nucleotides are depicted as follows: adenosine, A; guanosine, G; cytidine, C; uridine, U; purine, R;
pyrimidine, Y. tRNA (m
7
G46) methyltransferase transfers a methyl-group to the N7-atom of guanine
at position 46 in tRNA and forms 7-methylguanine. (B) The L-shaped structure of tRNA is presented.
The m
7
G46 forms a tertiary base pair with the C13-G22 base pair in the L-shaped tRNA structure. In
the stick model, only atoms are visible and charge is invisible.
There are some examples where m
7
G is found in positions other than at position 46 (Figure 2,
Table 1). Chloroplast tRNA
Leu
(UAG) from Chlamydomonas reinhardtii has m
7
G at position 36 in the
tRNA anticodon [16]. In animal mitochondria in which there is deviation from the universal genetic
code, m
7
G is present at position 34 in the anticodon of mitochondorial (mt) tRNA
Ser
(GCU) of starfish,
Asterina amurensis [17], and squid, Loligo bleekeri [18]. Furthermore, the D-arms of the mt
tRNA
Ser
(m
7
GCU) in starfish and squid have an unusual secondary structure, and the mt
tRNA
Ser
(m
7
GCU) recognizes not only the serine codons AGU and AGC but also the unusual serine
codons AGA and AGG. Thus, m
7
G can make base-pairing with the four basic bases of A, U, G, and
C [19]. With respect to m
7
G in archaeal tRNA, in 1991, Edmonds et al. reported that tRNA mixtures
from some archaea contain m
7
G nucleoside; however, the position(s) in tRNA has remained
unidentified [7]. More than twenty years later, It was demonstrated that the presence of the m
7
G
modification in a thermo-acidophilic archaeon, Thermooplasma acidophilum at the novel, irregular
position 49 in class II tRNA
Leu
[20]. However, the gene encoding the methyltransferase for m
7
G49 in
tRNA has not been identified thus far.
Table1. N7-methylguanosine methyltransferase in tRNA.
Domains of
life
Organisms Positions of m
7
G Enzyme names
Higher order
structure
References
eubacteria E. coli 46 TrmB monomer [51,56]
A. aeolicus 46 TrmB monomer [49,57,58]
T. thermophilus 46 TrmB ? [86]
B. subtilis 46 TrmB homodimer [50]
S. pneumoniae 46 TrmB homodimer
PDB:
1YZH
archaea T. acidophilum 49 (tRNA
Leu
(UAG)) ? ? [7,20]
T. neutrophilus ? ? ? [7]
eukaryote S. cerevisiae 46 Trm8/Trm82 heterodimer [46,52,66]
S. cerevisiae 5′ termini of pre-tRNA Ceg1p ? [24]
C. lagenarium 46 Aph1 ? [88]
Figure 1.
tRNA m
7
G46 methyltransferase methylates the N7-atom of guanine at position 46 in tRNA
and forms m
7
G46. (
A
) The secondary structure of tRNA is presented in cloverleaf form. Conserved
nucleotides are depicted as follows: adenosine, A; guanosine, G; cytidine, C; uridine, U; purine, R;
pyrimidine, Y. tRNA (m
7
G46) methyltransferase transfers a methyl-group to the N7-atom of guanine at
position 46 in tRNA and forms 7-methylguanine. (
B
) The L-shaped structure of tRNA is presented. The
m
7
G46 forms a tertiary base pair with the C13-G22 base pair in the L-shaped tRNA structure. In the
stick model, only atoms are visible and charge is invisible.
There are some examples where m
7
G is found in positions other than at position 46 (Figure 2,
Table 1). Chloroplast tRNA
Leu
(UAG) from Chlamydomonas reinhardtii has m
7
G at position 36 in
the tRNA anticodon [
16
]. In animal mitochondria in which there is deviation from the universal
genetic code, m
7
G is present at position 34 in the anticodon of mitochondorial (mt) tRNA
Ser
(GCU)
of starfish, Asterina amurensis [
17
], and squid, Loligo bleekeri [
18
]. Furthermore, the D-arms of
the mt tRNA
Ser
(m
7
GCU) in starfish and squid have an unusual secondary structure, and the mt
tRNA
Ser
(m
7
GCU) recognizes not only the serine codons AGU and AGC but also the unusual serine
codons AGA and AGG. Thus, m
7
G can make base-pairing with the four basic bases of A, U, G,
and C [
19
]. With respect to m
7
G in archaeal tRNA, in 1991, Edmonds et al. reported that tRNA
mixtures from some archaea contain m
7
G nucleoside; however, the position(s) in tRNA has remained
unidentified [
7
]. More than twenty years later, it was demonstrated that the presence of the m
7
G
modification in a thermo-acidophilic archaeon, Thermooplasma acidophilum at the novel, irregular
position 49 in class II tRNA
Leu
[
20
]. However, the gene encoding the methyltransferase for m
7
G49 in
tRNA has not been identified thus far.
Table 1. N7-methylguanosine methyltransferase in tRNA.
Domains of Life Organisms
Positions of m
7
G
Enzyme Names Higher Order Structure References
eubacteria E. coli 46 TrmB monomer [21,22]
A. aeolicus 46 TrmB monomer [23–25]
T. thermophilus 46 TrmB ? [26]
B. subtilis 46 TrmB homodimer [27]
S. pneumoniae 46 TrmB homodimer PDB: 1YZH
archaea T. acidophilum
49 (tRNA
Leu
(UAG))
? ? [7,20]
T. neutrophilus ? ? ? [7]
eukaryote S. cerevisiae 46 Trm8/Trm82 heterodimer [28–30]
S. cerevisiae 5
0
termini of pre-tRNA Ceg1p ? [31]
C. lagenarium 46 Aph1 ? [32]
human 46 METTL1/WDR4 ? [21,28]
C. reinhardtii
37 (chloroplast
tRNA
Leu
(UAG))
? ? [16]
A. amurensis 34 (mt tRNASer(GCU)) ? ? [17,19]
L. bleekeri 34 (mt tRNASer(GCU)) ? ? [18,19]
Unidentified m
7
G positions, enzymes, and higher order structures are indicated by question marks.
Int. J. Mol. Sci. 2018, 19, 4080 3 of 14
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 14
human 46 METTL1/WDR4 ? [46,51]
C. reinhardtii
37 (chloroplast
tRNA
Leu
(UAG))
? ? [16]
A. amurensis 34 (mt tRNASer(GCU)) ? ? [17,19]
L. bleekeri 34 (mt tRNASer(GCU)) ? ? [18,19]
Unidentified m
7
G positions, enzymes, and higher order structures are indicated by question marks.
The 7-methylguanosine modifications occur not only in tRNA, but also in other RNA species
such as mRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), and small nucleolar RNA
(snoRNA). The 5′ terminus of eukaryotic mRNA is blocked by m
7
G
5′
ppp
5′
N cap structure [21,22], and
gene of cap-m
7
G methyltransferase (Abd1) is essential in Saccharomyces cerevisiae [23]. Surprisingly,
recent work has detected a cap structure in tRNA. In yeast, an m
7
G cap structure is found at the 5′
termini of pre-tRNA bearing 5′ leader sequences (Figure 2). The capped pre-tRNAs accumulate due
to inhibition of 5′ exonucleases activities and protect pre-tRNAs from 5′-exonucleolytic degradation
during maturation [24]. 7-methylguanosine is observed in 16S rRNA of aminoglycoside-producing
Actinobacteria, including Streptomyces tenebrarius and Micromonospora purpurea. The m
7
G
modification is at position G1405 in the 16S rRNA and has an aminoglycoside resistance activity [25–
27].
Figure 2. Positions of the m
7
G modification in tRNA. The numbers in circles indicate the positions of
m
7
G in tRNA.
Since Holley et al. determined the sequence of yeast tRNA
Ala
in 1965 [28], various tRNA
sequences have been reported, and the presences of modified nucleosides in tRNA have been
revealed. Also, the technical method of m
7
G detection has a history as well as tRNA sequencing.
Initially, Wintermeyer and Zachau described a specific chemical method in which m
7
G detection is
achieved via aniline-induced cleavage of the tRNA strand by β-elimination after additional treatment
under alkaline conditions or after its reduction by sodium borohydride (NaBH
4) in tRNA [29,30].
Both reduced m
7
G and its degradation products are susceptible to hydrolysis of the N-glycoside bond
with subsequent chain scission by the β-elimination. By combining the aniline cleavage method and
the Donis-Keller-method, which uses ribonucleases [31,32], it is possible to identify the position of
m
7
G in tRNA. These methods are utilized for m
7
G detection not only in tRNA but also in rRNA [33].
In the Donis-Keller-method, RNsase T1 is used for detection of guanosine position. Although RNase
T1 specifically digests the phosphodiester bond of guanosine-3′ phosphate in ribonucleic acid and
ribonucleotide, the 7-methyl modification of guanosine prevents RNase T1 cleavage. Additionally, in
pre-tRNA containing m
7
G at the 5′-end of the acceptor stem, the m
7
G modified nucleoside absolutely
prevents cleavage by M1 RNA, the catalytic RNA subunit of RNase P [34]. Inhibition by m
7
G is
Figure 2.
Positions of the m
7
G modification in tRNA. The numbers in circles indicate the positions of
m
7
G in tRNA.
The 7-methylguanosine modifications occur not only in tRNA, but also in other RNA species
such as mRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), and small nucleolar RNA
(snoRNA). The 5
0
terminus of eukaryotic mRNA is blocked by m
7
G
5
0
ppp
5
0
N cap structure [
33
,
34
], and
gene of cap-m
7
G methyltransferase (Abd1) is essential in Saccharomyces cerevisiae [
35
]. Surprisingly,
recent work has detected a cap structure in tRNA. In yeast, an m
7
G cap structure is found at the 5
0
termini of pre-tRNA bearing 5
0
leader sequences (Figure 2). The capped pre-tRNAs accumulate due
to inhibition of 5
0
exonucleases activities and protect pre-tRNAs from 5
0
-exonucleolytic degradation
during maturation [
31
]. 7-methylguanosine is observed in 16S rRNA of aminoglycoside-producing
Actinobacteria, including Streptomyces tenebrarius and Micromonospora purpurea. The m
7
G modification
is at position G1405 in the 16S rRNA and has an aminoglycoside resistance activity [36–38].
Since Holley et al. determined the sequence of yeast tRNA
Ala
in 1965 [
39
], various tRNA sequences
have been reported, and the presences of modified nucleosides in tRNA have been revealed. Also, the
technical method of m
7
G detection has a history as well as tRNA sequencing. Initially, Wintermeyer and
Zachau described a specific chemical method in which m
7
G detection is achieved via aniline-induced
cleavage of the tRNA strand by
β
-elimination after additional treatment under alkaline conditions
or after its reduction by sodium borohydride (NaBH
4
) in tRNA [
40
,
41
]. Both reduced m
7
G and its
degradation products are susceptible to hydrolysis of the N-glycoside bond with subsequent chain
scission by the
β
-elimination. By combining the aniline cleavage method and the Donis-Keller-method,
which uses ribonucleases [
42
,
43
], it is possible to identify the position of m
7
G in tRNA. These methods
are utilized for m
7
G detection not only in tRNA but also in rRNA [
44
]. In the Donis-Keller-method,
RNsase T1 is used for detection of guanosine position. Although RNase T1 specifically digests the
phosphodiester bond of guanosine-3
0
phosphate in ribonucleic acid and ribonucleotide, the 7-methyl
modification of guanosine prevents RNase T1 cleavage. Additionally, in pre-tRNA containing m
7
G
at the 5
0
-end of the acceptor stem, the m
7
G modified nucleoside absolutely prevents cleavage by M1
RNA, the catalytic RNA subunit of RNase P [
45
]. Inhibition by m
7
G is thought to be because the
approach of positive magnesium ion as a cleavage agent becomes impossible due to the positive charge
of m
7
G. In addition, antibodies which specifically target N
6
-methyladenine (m
6
A) and m
7
G were
prepared by immunization of rabbits with nucleoside conjugates of bovine serum albumin (m
6
A-BSA,
m
7
G-BSA) [
46
]. Both the anti-m
7
G and anti-m
6
A antibody adsorbents became a tool for fractionation
of oligonucleotides and nucleic acids. Currently, not only the m
7
G modification but also a variety of
other modified nucleosides can be detected more quickly and accurately by mass spectrometry or high
performance liquid chromatography [
47
]. Moreover, only recently, Motorin and co-authors reported a
Int. J. Mol. Sci. 2018, 19, 4080 4 of 14
deep sequencing method named AlkAniline-Seq for the detection of m
7
G in RNA at single nucleotide
resolution. AlkAniline-Seq is exploited the generation of abasic sites by alkaline hydrolysis and aniline
cleavage. The method allows for sensitive m
7
G detection of total RNA from cells [48].
The enzymatic activity of tRNA (m
7
G46) methyltransferase was initially confirmed in cell extracts
from Escherichia coli [
49
] and has been purified more than 1000-fold [
50
]. Enzymatic activities have also
been detected from Salmonella typhimurium [
51
,
52
], Thermus flavus [
53
], Xenopus laevis [
54
], humans [
55
],
and plants [
9
]. The m
7
G46 modification is generated by tRNA (m
7
G46) methyltransferase (tRNA
(guanine-7-)-methyltransferase, EC 2.1.1.33; TrMet (m
7
G46)) [
3
,
56
] The gene encoding tRNA (m
7
G46)
methyltransferase was first identified in yeast and was shown to be composed of two protein subunits
Trm8 and Trm82 encoded by YDL201w and YDR165w, respectively [
28
]. Although Trm8 is the catalytic
subunit, formation of a complex with Trm82 is required for the enzymatic activity [
57
]. Following this
report, eubacterial genes have also been identified as trmB, whose classical name is yggh, in E. coli [
58
],
Aquifex aeolicus [23], and Bacillus subtilis [27] (Table 1).
In this review, information of tRNA m
7
G modifications and tRNA m
7
G methyltransferases since
m
7
G was discovered in tRNA is summarized, and the differences in reaction mechanism between
tRNA m
7
G methyltransferase and rRNA or mRNA m
7
G methylation enzyme are discussed.
2. Structural Analyses and Catalytic Mechanisms of m
7
G Methyltransferases
Structural studies of tRNA modification enzymes can be informative both for the specificity and
catalytic mechanism of the enzymes. Furthermore, the combination of structural and biochemical
analysis data allows comparison of reaction mechanisms from different species. This makes it
possible to infer information about molecular evolution. The crystal structures of tRNA (m
7
G46)
methyltransferase from B. subtilis [
27
], E. coli [
21
], Streptococcus pneumoniae (PDB: 1YZH), and
S. cerevisiae [
29
] have been reported (Table 1). X-ray crystallographies of TrmB and Trm8 have revealed
classic class I AdoMet-dependent methyltransferase structures. These AdoMet-dependent tRNA
MTases belong to two protein super families, which are structurally and phylogenetically unrelated,
namely, the Rossmann fold MTases (RFM) and SPOUT MTases (SpoU and TrmD) [
59
]. SPOUT MTases
have a deep trefoil knot structure which forms the catalytic site and the cofactor-binding pocket [
59
–
61
].
TrmB and Trm8 belong to the RFM family of MTases [29,58].
2.1. Eubacterial tRNA m
7
G46 Methyltransferases (TrmB)
Eubacterial TrmB exists as either a single subunit or a homodimer. TrmB of B. subtilis and
S. pneumoniae has a dimeric structure both in solution and in the crystal form. However, dimerization
does not seem to be a common feature of the enzymes, as the interface is not conserved and the TrmB
enzymes of E. coli and A. aeolicus are monomeric. Analysis of TrmB activity using mutant proteins
based on bioinformatic studies has revealed residues important for function [
22
]. The thermophilic
TrmB has a longer C-terminal region compared to the methophilic TrmB [
23
,
24
] (Figure 3B). It has
been reported that the C-terminal region is required for protein stability at high temperatures and
contributes to the selection of the precise guanine nucleotide (i.e., G46) to be modified [
24
]. Alanine
substitution of Arg287 in the long C-terminal region considerably reduces the methyltransfer activity.
Thus, a part of the C-terminal region may make contact with tRNA. In contrast, the methophilic
TrmB proteins from E. coli and B. subtilis have a long N-terminal region, and it has been shown that
Arg26 in E. coli TrmB is involved in activity [
22
]. Furthermore, when Asp133 is replaced by alanine or
asparagine in A. aeolicus TrmB, the methyltransfer activity is completely lost [
24
]. The aspartic acid
residue is highly conserved in both TrmB and Trm8. Therefore, the Asp133 of A. aeolicus TrmB is
considered as the catalytic center. Furthermore, in a docking model of guanine and the Trm8–Trm82
complex, the corresponding aspartic acid has the same positional relationship [29]. For this reason, it
is suggested that Asp133 of A. aeolicus TrmB captures the G46 base of tRNA. Mutagenesis study has
shown that Asp133 and several amino acid residues may contribute to AdoMet binding (Figure 4A).
Taken together, it has been proposed a hypothetical mechanism for TrmB in which the carboxyl group
Int. J. Mol. Sci. 2018, 19, 4080 5 of 14
of Asp133 captures the proton of N–H of the guanine base and the N7 atom of the guanine base itself
attacks the methyl group in AdoMet [
25
] (Figure 4B). A hypothetical catalytic mechanism for TrmI,
which is a methyltransferase for the N1 atom of adenosine at position 58 in tRNA, has also been
proposed [
62
]. In this mechanism, the N1 atom causes a nucleophilic attack on the methyl group of
AdoMet. Since the reactivity of nitrogen atoms in the bases is generally higher than the reactivity of
carbon and oxygen atoms [
56
], in some methyl group transfer reactions, the nitrogen atom itself seems
to directly attack the methyl group of AdoMet.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 14
asparagine in A. aeolicus TrmB, the methyltransfer activity is completely lost [57]. The aspartic acid
residue is highly conserved in both TrmB and Trm8. Therefore, the Asp133 of A. aeolicus TrmB is
considered as the catalytic center. Furthermore, in a docking model of guanine and the Trm8–Trm82
complex, the corresponding aspartic acid has the same positional relationship [52]. For this reason, it
is suggested that Asp133 of A. aeolicus TrmB captures the G46 base of tRNA. Mutagenesis study has
shown that Asp133 and several amino acid residues may contribute to AdoMet binding (Figure 4A).
Taken together, it has been proposed a hypothetical mechanism for TrmB in which the carboxyl
group of Asp133 captures the proton of N–H of the guanine base and the N7 atom of the guanine
base itself attacks the methyl group in AdoMet [58] (Figure 4B). A hypothetical catalytic mechanism
for TrmI, which is a methyltransferase for the N1 atom of adenosine at position 58 in tRNA, has also
been proposed [59]. In this mechanism, the N1 atom causes a nucleophilic attack on the methyl group
of AdoMet. Since the reactivity of nitrogen atoms in the bases is generally higher than the reactivity
of carbon and oxygen atoms [45], in some methyl group transfer reactions, the nitrogen atom itself
seems to directly attack the methyl group of AdoMet.
Figure 3. Comparison of thermophilic and methophilic TrmB. (A) Sequence alignment of TrmB.
Conserved regions are highlighted in three colored squares (red, green, blue). Asp133 is indicated by
an asterisk. (B) Thermophilic and mesophilic TrmB proteins are illustrated schematically. The three
colored regions correspond to the amino acid sequences in panel A. Thermophilic TrmB has a distinct
long C-terminal region. Asp133 is highlighted with an arrow, and the red D corresponds to the Asp133.
Figure 3.
Comparison of thermophilic and methophilic TrmB. (
A
) Sequence alignment of TrmB.
Conserved regions are highlighted in three colored squares (red, green, blue). Asp133 is indicated by
an asterisk. (
B
) Thermophilic and mesophilic TrmB proteins are illustrated schematically. The three
colored regions correspond to the amino acid sequences in panel A. Thermophilic TrmB has a distinct
long C-terminal region. Asp133 is highlighted with an arrow, and the red D corresponds to the Asp133.