Transfer RNA Synthesis and Regulation: Advanced Article

Transfer RNA Synthesis
and Regulation
Hiroyuki Hori, Ehime University, Ehime, Japan
Chie Tomikawa, Ehime University, Ehime, Japan
Akira Hirata, Ehime University, Ehime, Japan
Yukimatsu Toh, AIST, Tsukuba, Japan
Kozo Tomita, AIST, Tsukuba, Japan
Takuya Ueda, University of Tokyo, Kashiwa, Japan
Kimitsuna Watanabe, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Advanced article
Article Contents
. Introduction
. Prokaryotic Systems
. Eukaryotic Systems
. Synthesis of Mature tRNA
. Summary
Online posting date: 15th September 2014
Based in part on the previous version of this eLS article Transfer RNA
Synthesis and Regulation (2009) by Yukimatsu Toh, Hiroyuki Hori,
Kozo Tomita, Takuya Ueda and Kimitsuna Watanabe.
Transfer ribonucleic acid (tRNA), which is primarily transcribed from tRNA genes by RNA polymerase, matures via
several steps: processing, splicing, CCA addition and posttranscriptional modifications. Primary transcripts of
tRNA genes contain extra 5 and 3 sequences, which are
removed by a set of nucleases. In addition, some primary
transcripts contain introns, which are spliced out by specific endonucleases or in self-splicing reactions. The ligation of exons generally requires a tRNA ligase. In some
species, the CCA sequences present at the 3-termini of all
mature tRNAs are not encoded in the tRNA genes, but are
added post-transcriptionally by a CCA-adding enzyme. All
mature tRNA molecules contain modified nucleotides,
generated by specific tRNA modification enzymes or
guide RNA systems. These modified nucleotides are
involved in stabilisation of tRNA structure, decoding,
tRNA quality control, regulation of subcellular localisation of tRNAs and immune responses against infectious
organisms.
Introduction
To ensure ecient translation, the size and composition of
the intracellular transfer ribonucleic acid (tRNA) pool is
regulated at several stages of RNA synthesis and degradation. Codons residing in the open reading frames
eLS subject area: Biochemistry
How to cite:
Hori, Hiroyuki; Tomikawa, Chie; Hirata, Akira; Toh, Yukimatsu; Tomita,
Kozo; Ueda, Takuya; and Watanabe, Kimitsuna (September 2014)
Transfer RNA Synthesis and Regulation. In: eLS. John Wiley & Sons, Ltd:
Chichester.
DOI: 10.1002/9780470015902.a0000529.pub3
(ORFs) of protein-encoding genes are not equally utilised.

Such codon biases may reect nucleotide selection resulting from directional mutation pressure during evolution.
For example, a genome with high GC content will preferentially contain codons containing G or C in its ORFs.
Correlations between codon preference and genomic GC
content are found mainly in highly expressed proteinencoding genes, but are not necessarily observed in the
ORFs of genes expressed at lower levels. The concentrations of tRNA species in cells are closely related to codon
bias in the ORFs of protein-encoding genes. For example,
in Micrococcus luteus, whose genomic GC content is
approximately 75%, a correlation between codon usage
and the content of the corresponding tRNA species is
clearly evident (Figure 1). High expression of a plasmidencoded gene containing a minor codon occasionally
causes ribosome pausing; therefore, it is likely that regulation of the content of various tRNA species in cells is
involved in the maintenance of the translational rate of
messenger RNAs (mRNAs) on ribosomes. Codons corresponding to abundant tRNAs are designated as optimal
codons (Ikemura, 1982). Thus, control of tRNA abundance is one of the fundamental mechanisms for achieving
economical protein synthesis. See also: Codon Usage in
Molecular Evolution; Genetic Code: Introduction;
Transfer RNA
The relative amounts of tRNA species are regulated by
several factors, such as copy number of tRNA genes,
transcriptional activity and RNA degradation. In addition, the environment surrounding the cells, including
nutrient content and the presence of various stresses, also
inuences the balance of individual tRNAs. See also: Gene
Expression: Decoding and Accuracy of Translation
Maturation of tRNA molecules usually requires several
steps. Primary transcripts of tRNA genes contain extra 5
and 3 sequences, which are removed by a set of nucleases.
Introns are found in tRNA genes in eukaryotes, archaea,
chloroplasts, plant mitochondria, and some eubacteria.
eLS & 2014, John Wiley & Sons, Ltd. www.els.net
2
100
70
Amount of tRNA
Amino acid
Codon
Anticodon
89
CAG
CUG
CUY
100
GAG
1 UUG
0 CUA
0 UUA
AUY
100
0 AUA
100
GUG
GUY
84
Leu
lle
Val
CCG
67
CCY
Pro
>1 CCA
100
52
ACY
ACG
>1 ACA
50
AAG
0 AAA
100
100
25
100
CAC
68
100
66
GGG
100
*UGG ND
Thr
GGU
CGU
*UGU ND
Ala
GGC
CGC
100
39
*UGC ND
CUU
*UUU ND
100
CGY/A
ICG
CGG Arg CCG 8
3 AGG
CCU ND
*UCC ND
0 AGA
100
Lys
100
GAU
CGG
Anticodon
5 GCA
*CAU ND
GAC
GCY
GCG
100
Amount of tRNA
Amino acid
Codon
*UAC ND
GUA
100
CAA ND
*UAG ND
*UAA ND
Codon usage
Transfer RNA Synthesis and Regulation
Codon usage
12
GGY
GGG
2 GGA
GCC
Gly
100
CCC
*UCC ND
100
85
Figure 1 Relative codon usages and tRNA levels in Micrococcus luteus. Codon usages and amounts of isoacceptor tRNAs are shown relative to the most abundant species (defined as 100). ND, not detected;
indicates a modified nucleotide; Y refers to U or C and I is inosine. Reprinted with permission from Kano A et al. (1991) & Elsevier.
In general, introns are spliced out by a tRNA-specic

splicing endonuclease, and the two resultant RNA fragments are joined by RNA ligase. In the case of introns in
eubacterial tRNAs, a self-splicing reaction is involved in
intron removal and exon ligation. The CCA sequences at
the 3-terminal of all tRNAs are completely conserved
characterised to date. In some species, these sequences are
not encoded in the tRNA genes, but are introduced posttranscriptionally by a CCA-adding enzyme. All mature
tRNA molecules contain modied nucleotides, which are
involved in the stabilisation of structure and in the
decoding process. In addition, modied nucleotides are
involved in tRNA quality control systems, regulation of
subcellular localisation of tRNAs, infection and immune
responses. These modied nucleotides are converted from
canonical nucleotides by specic tRNA modication
enzymes or guide RNA systems after transcription of the
precursor molecules. See also: Transfer RNA Modication; Transfer RNA Structure
Prokaryotic Systems
Gene-dosage effects
All tRNA genes in the Escherichia coli genome have been
identied and mapped (Komine et al., 1990; Blattner et al.,
1997). Escherichia coli has 86 tRNA genes corresponding
to 46 tRNA species, and the copy number for each tRNA
species ranges from one to four. tRNA genes corresponding to codons that frequently appear in ORFs of highly
expressed genes are always present in four copies, whereas
codons with moderate usage are translated by tRNAs
encoded by genes with two or three copies. By contrast,
tRNAs corresponding to rare codons are encoded by a
single gene. Gene dosage is thus clearly involved in the
regulation of tRNA concentrations. In addition to this
gene-dosage eect, it is likely that another mechanism,
promoter activity, simultaneously participates in the control of tRNA concentration.
Promoter activity
The amount of a given tRNA is not perfectly commensurate with its copy number. For example, in E. coli cells,
isoleucine tRNA corresponding to codons AUC and AUU
is present at a 20-fold higher concentration than the isoacceptor tRNA specic to the rare codon AUA, although
the respective copy numbers are three for the common
tRNA and one for the rare one. This discrepancy can be
explained by the involvement of promoter activity in regulation of tRNA contents. In E. coli, 40 tRNA genes are
found in operons and are thus primarily produced in
transcriptional units under the control of single promoters.
Ten tRNA genes are present in the spacer regions of
ribosomal RNA (rRNA) operons, and six tRNA operons
contain protein genes, such as the EF-Tu gene. Because
EF-Tu and rRNA are highly expressed in cells, these
promoters have strong activity; as a result, the anking

tRNAs are also thought to be eciently transcribed. Thus,
it is evident that promoter activity also participates in the
control of tRNA contents.
Degradation processes
Another mechanism for control of tRNA concentrations
was clearly demonstrated by extensive analysis of intracellular tRNA levels in Mycoplasma capricolum, which has
a highly AT-biased and compact genome (Muto et al.,
1990). In protein-encoding genes of M. capricolum, codons
terminating with U or A are frequently utilised. Almost all
tRNA genes in this bacteriums genome are encoded as
single-copy genes: 30 tRNA genes generate 29 tRNA species. However, the concentration of each tRNA species
diers greatly, ruling out the possibility that copy number
is solely responsible for regulating the tRNA concentration. Although the mechanism of tRNA gene expression
has not been systematically analysed, the following
observation suggests that tRNA degradation is also
involved in control of tRNA content: On the M. capricolum
genome, 22 of 30 tRNA genes are organised in polycistronic operons containing only tRNA genes, in contrast
to the case of E. coli. Although multiple tRNAs are transcribed in transcriptional units, the concentrations of
mature tRNA species dier from one to another. Hence,
the lifetime of a particular tRNA is related to the abundance of the tRNA molecule. Furthermore, the authors
recently found that in the thermophilic eubacterium
Thermus thermophilus, hypomodication at multiple sites
in tRNA due to disruption of one of the modication
enzymes promotes degradation of tRNAPhe and tRNALys
at high temperatures (Tomikawa et al., 2010). Thus, tRNA
modications are involved in regulation of tRNA halflives. Although several nucleases from various species have
been puried and characterised, the molecular mechanism
of tRNA degradation in prokaryotes remains unknown.
Environment and tRNA concentration

effects
Protein synthesis is delicately modulated in response to the
environment surrounding the cell. Amino acid starvation
causes arrest of protein synthesis; this arrest is mediated by
guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp). This process, called stringent control, prevents unnecessary consumption of energy.
Expression of most bacterial tRNA genes is under stringent
control; consequently, transcription of these genes is
inhibited under conditions of amino acid starvation.
ppGpp and pppGpp are synthesised on the ribosome from
guanosine triphosphate (GTP) and adenosine triphosphate (ATP) by a stringency factor in response to elevated
concentrations of uncharged tRNAs in amino acid-starved
cells. ppGpp or pppGpp probably interacts with RNA
polymerase to inhibit tRNA transcription, resulting in a
deciency of tRNA and arrest of protein synthesis. A 7-bp
GC-rich sequence, which may be responsible for stringent

control, is found between the 210 element of the promoter
and the transcription start site of E. coli tRNA genes.
Growth rate may also inuence the balance of tRNA
concentrations in E. coli. At high growth rates, tRNAs
specic for optimal codons become relatively more abundant than those corresponding to minor codons. Although
the molecular mechanism involved in this type of control is
totally unknown, such a correlation is logically consistent
with the requirements for economical protein synthesis.
See also: Translational Components in Prokaryotes:
Genetics and Regulation of Ribosomes
Physiological conditions, degradation,

subcellular localisation, stress responses and
tissue specificity
Eukaryotic Systems
Transcriptional control
The relative amounts of tRNA molecules in yeast have
been thoroughly analysed, and the tRNA concentrations
correlate well with the usage of cognate codons. Owing to
recent large-scale genome analyses, information about
eukaryotic tRNA genes has increased rapidly over the past
15 years (Lowe and Eddy, 1997; Juhling et al., 2009; Abe
et al., 2009). The molecular mechanisms responsible for the
regulation of tRNA concentrations remain to be elucidated, but transcription has been implicated in these
processes.
The transcription of eukaryotic tRNA genes is more
complex than that of bacterial tRNA. The transcriptional
machinery containing RNA polymerase III, which is
responsible for transcribing eukaryotic tRNA genes,
requires short conserved sequences, called the A and B
boxes, downstream of the transcriptional start site (Geiduschek and Tocchini-Valentine, 1988). These boxes correspond to the coding regions for the D- and T-arms in
tRNA, respectively. These two short stretches are essential
for the binding of a transcription complex comprising two
transcriptional factors, TFIIIB and TFIIIC, as well as for
initiation of transcription. In addition to the A and B
boxes, other regions contribute to the eciency of transcription. In the 5 upstream anking regions, elements
inuencing transcriptional eciency have been identied
in both insects and vertebrates. These elements, which act
either positively or negatively, are likely to be related to the
control of tRNA expression. Additionally, 3 anking
elements also inuence transcriptional activity. These 5
and 3 anking elements are important for binding of
specic transcription factors that control particular
tRNAs. However, the manner in which such anking
sequences are involved in the regulation of tRNA concentrations remains unknown.
In addition, the Maf1 protein inhibits the transcription
of tRNA by acting as a repressor of RNA polymerase III
(Pol III) (Ciesla and Boguta, 2008). This protein was
initially identied in yeast, and later shown to be evolutionarily conserved: Maf1 orthologues also restrain transcription in mouse and human cells. Transcription
4
regulation by Maf1 in yeast is important for the switch

between fermentation and respiration. Under respiratory
conditions, Maf1 is activated by dephosphorylation and
imported into the nucleus, where it inhibits tRNA transcription. The transition from a nonfermentable carbon
source to glucose induces Maf1 phosphorylation and its
relocation to the cytoplasm. The absence of Maf1-mediated control of tRNA synthesis impairs cell viability on
nonfermentable carbon sources (Ciesla et al., 2007).
See also: RNA Polymerases: Subunits and Functional
Domains; Transcriptional Gene Regulation in Eukaryotes
As in the prokaryotic system, regulation of mature tRNA

concentration occurs in eukaryotes. Removal of CCA,
catalysed by a specic nuclease, blocks the attachment of
cognate amino acids. This process prevents aminoacyltRNA synthetase from abortive aminoacylation, thereby
saving ATP. Furthermore, some modied nucleotides
aect tRNA degradation systems. The m1A58 modication is essential for cell viability (Table 1). The precursor
initiator tRNAMet without m1A58 modication is polyadenylated by the so-called TRAMP complex, and then
degraded by Rrp6 and the nuclear exosome (Kadaba et al.,
2004). Furthermore, a yeast double mutant-strain in which
both tRNA (m7G46) methyltransferase and tRNA
(m5C34, 40, 48, 49) methyltransferase genes are disrupted,
exhibits a severe growth defect and a shortened half-life of
tRNAVal (Table 1). The hypomodied tRNAs are aggressively degraded by the rapid tRNA decay system (Chernyakov et al., 2008). Moreover, subcellular localisation,
including transport from nucleus to cytoplasm and import
from cytoplasm to organelles regulates tRNA populations
in eukaryotic cells. The main pathway of transport from
nucleus to cytoplasm in yeast is a Ran-GTP-dependent
system in which maturation of tRNA is monitored by
aminoacylation (Lund and Dahlberg, 1998). In Leishmania
tarentolae, wobble modications (mcm5s2U34 and
mcm5Um34) of tRNAGlu and tRNAGln control subcellular
localisation of these tRNA species (Kaneko et al., 2003).
Several stresses induce cleavage of tRNA. For example,
tRNA cleavage occurs during amino acid starvation in
Tetrahymena (Lee and Collins, 2005), as well as under
oxidative stress in yeast, Arabidopsis, and human
(Thompson et al., 2008). Furthermore, initiator tRNAMetspecic cleavage and accumulation of initiator tRNAMet
has been observed in the nucleus of heat-shocked HeLa
cells (Watanabe et al., 2013). The cleaved tRNA fragments
might play biological roles, for example, in the inhibition of
translation (Thompson and Parker, 2009).
In the silk gland of the silkworm, only three tRNAs are
highly expressed during the synthesis of the silk protein,
which consists mainly of alanine, glycine and serine. The
tRNAs corresponding to these three amino acids account
Table 1 Role of modied nucleosides in transfer ribonucleic acid (tRNA)

Location
Modied nucleosides
Function
References
Wobble position
Q (queuosine)
Promotion of pairing with C and U of codon

third letter
Pairing with C, A and U of codon third letter
Pairing with G and A of codon third letter
Alteration of amino acid- specicity of
tRNAIle and codon recognition (from AUG
to AUA, AUU and AUC)
Expanding or restricting the decoding
capacity of tRNA
(Okada et al., 1979; Urbonavi0ius et al., 2001)
I (inosine)
f5C (5-formylcytidine)
L (k2C, lysidine) and agm2C(agmatidine)
Modied uridines (cmo5U, cmnm5s2U,

mnm5s2U, tm5U, tm5s2U)
2-Thiolated uridines
2-O-methylated pyrimidine
Anticodon arm
Stabilisation of C3-endo form of ribose,

restriction or promotion of codon
anticodon base-pairing, control of
subcellular localisation, promotion of
codonanticodon base pairing, control of
subcellular localisation
Stabilisation of C3-endo form of ribose and
reinforcement of codonanticodon
interaction
Prevention of frameshift error
M1G37
Prevention of mischarging of tRNAAsp by

arginyl-tRNA synthetase, maintenance of
reading frame
Hypermodied purine37 (yW, i6A, t6A,

ms2i6A, ms2t6A, cyclic t6A)
Stabilisation of codonanticodon
interaction or U-turn structure in the
anticodon loop by reinforcing stacking,
maintenance of reading frame
Stabilisation of the anticodon structure
2-O-methylated nucleosides 32, 34, 39
C38, C39, C40
m5C34, m5C40
Suppression of misreading by tRNAHis,

regulation of translation of His and Ile-Val
operons
Deletion of these nucleosides together with
m5C48, m5C49 and m7G46 results in severe
growth defect of yeast
(Pintard et al., 2002; Guy et al., 2012; Clouet

dOrval et al., 2001; Bortolin et al., 2003; Singh
et al., 2004; Joardar et al., 2011; Ben tez-Paez
et al., 2010; Liu et al., 2013)
(Urbonavi0ius et al., 2001; Bjork et al., 1989;
Farabaugh and Bjork, 1999)
(Urbonavi0ius et al., 2001; Perret et al., 1990;
Bjork et al., 2001; Christian et al., 2004; Brule
et al., 2004; Goto-Ito et al., 2009; Paris et al.,
2013)
(Noma et al., 2006; Caillet and Droogmans,
1988; Warner et al., 2000; Yarian et al., 2002;
Miyauchi et al., 2013)
(Pintard et al., 2002; Guy et al., 2012; Clouet
dOrval et al., 2001; Bortolin et al., 2003; Singh
et al., 2004; Purta et al., 2006)
(Hur and Stroud, 2007; Behm-Ansmant et al.,
2004; Kammen et al., 1988)
(Alexandrov et al., 2006; Brzezicha et al., 2006;
Auxilien et al., 2007; Kuratani et al., 2010)
(continued )
Modied G37 and A37
(Wolf et al., 2002)

(Takemoto et al., 2008)
(Muramatsu et al., 1988; Soma et al., 2003;
Ikeuchi et al., 2005, 2010; Mandal et al., 2010;
Terasaka et al., 2011; Osawa et al., 2011)
(Takai and Yokoyama, 2003; Weixlbaumer
et al., 2007; Yasukawa et al., 2001; Kirino
et al., 2004; Suzuki et al., 2002)
(Takai and Yokoyama, 2013; Ikeuchi et al.,
2006; Kawai et al., 1992)
Table 1
Continued
Three-dimensional
core
Modied nucleosides
Function
References
s U8, 9
Function unknown
(Lauhon et al., 2004)
m2G and m22G6, 7, 10, 26, 27
Stabilisation of tRNA structure
D16, 17, 20
Function unknown
Archaeosine15
Gm18
Stabilisation of tRNA structure

Interaction with C55; supression of
frameshift errors; suppression of immune
response via TLR7
m7G46
Formation of tertiary base pair with C13G22 in the D stem
m5C48, m5C49
Deletion of these nucleosides together with

m5C34, m5C40 and m7G46 results in severe
growth defect of yeast
Stabilisation of the interaction between
T- and D-loops
(Roovers et al., 2012; Fislage et al., 2012;

Menezes et al., 2011; Armengaud et al., 2004;
Urbonavicius et al., 2006; Hopper et al., 1982;
Constantinesco et al., 1998; Ihsanawati et al.,
2008; Awai et al., 2009; Awai et al., 2011)
(Bishop et al., 2002; Xing et al., 2002; Yu et al.,
2011)
(Watanabe et al., 2001; Ishitani et al., 2003)
(Urbonavicius et al., 2002; Persson et al., 1997;
Hori et al., 1998; Cavaille et al., 1999; Jockel
et al., 2012; Gehrig et al., 2012; Ochi et al.,
2013; De Bie et al., 2003; Okamoto et al., 2004)
(Hur and Stroud, 2007; De Bie et al., 2003;
Okamoto et al., 2004; Alexandrov et al., 2002;
Tomikawa et al., 2008; Leulliot et al., 2008)
(Alexandrov et al., 2006; Brzezicha et al., 2006;
Auxilien et al., 2007; Kuratani et al., 2010)
T54
s2T54
C55
Stabilisation of the interaction between

T- and D-loops; thermostability of tRNA;
increases thermostability of tRNA
Interaction with Gm18; suppression of
frameshift errors
m1A58
Necessary for growth at high temperature;

prolongs half-life of initiator tRNAMet
necessary for cell viability
A64 with 2-ribosyl phosphate
Distinguishing elongator from initiator

tRNAMet
(Urbonavicius et al., 2002; Gu et al., 1996;

Watanabe et al., 1974; Watanabe et al., 1976;
Shigi et al., 2006; Urbonavicius et al., 2005;
Nishimasu et al., 2009; Yamagami et al., 2012;
Hamdane et al., 2012)
(Awai et al., 2009; Watanabe et al., 1974;
Watanabe et al., 1976; Shigi et al., 2006)
(Urbonavicius et al., 2002; Cortese et al., 1974;
Nurse et al., 1995; Roovers et al., 2006; Muller
et al., 2007; Gurha and Gupta, 2008; Becker
et al., 1997; Hoang and Ferre-DAmare,2001;
Pan et al., 2003; Ishida et al., 2011)
(Anderson et al., 1998; Anderson et al., 2000;
Chujo and Suzuki, 2012; Droogmans et al.,
2003; Roovers et al., 2004; Guelorget et al.,
2010)
(Astrom and Bystrom,1994)
Location
for approximately 90% of all tRNA in this tissue, whereas

no such enrichment is observed in other organs. Such tissue-specic tRNA synthesis appears to be mediated at the
transcriptional level.
Synthesis of Mature tRNA

tRNA processing
The primary transcript of a tRNA gene usually contains
extra 5 and 3 sequences that are eventually removed by
various nucleases. In E. coli, the 3 trailer sequence of the
primary transcript is trimmed by several nucleases before 5
processing by RNase P. Multiple exoribonucleases,
including RNase D, RNase BN, RNase T, RNase PH,
RNase II, and polynucleotide phosphorylase, are involved
in the maturation of the 3 end of tRNA molecules (Li and
Deutscher, 1996). RNase PH trims o the 3 trailer extension by phospholysis, using an inorganic phosphate bound
to its active site. The 3 tail is trimmed because it is
accommodated into the inner surface of a ring, formed by
hexameric RNase PH. In eubacteris, in addition to the
exonucleases, an endonuclease termed RNase Z also plays
a role in maturation of the 3 end of tRNA. RNase Z cleaves
the phosphodiester bond located at the 3 site of the discriminator nucleotide, leaving a 3 hydroxyl group on its 3
end. RNase Z has been identied in all three domains of life
(Spath et al., 2007). Although many nucleases have been
shown to be involved in the processing of the 3 end of
precursor tRNA molecules, the detailed mechanism of 3end processing by these nucleases remains obscure.
In contrast to the complicated process 3 maturation, 5
processing is conducted by a single endonuclease, RNase P,
which is a complex of protein and RNA. Eubacterial
RNase P recognises external guide sequences (EGS) located in the acceptor stems of tRNA molecules. The RNA
component of RNase P itself is capable of catalysing a
cleavage reaction at the 5 terminus of precursor tRNA. In
eukarya and archaea, RNase P is composed of 410 proteins and an RNA molecule structurally similar to the
eubacterial RNase P RNA. However, neither the RNA
molecule nor the protein subunit is capable of catalysing
the cleavage of the 5 leaders of precursor tRNAs (Kirsebom, 2007). In human mitochondria, a protein complex
composed of three kinds of proteins, MRPP1, -2, and -3
(mitochondrial RNase P proteins 1, 2, and 3), is capable of
removing the 5 leader from precursor tRNA. The processing complex does not contain an RNA molecule
(Holzmann et al., 2008; Walker and Engelke, 2008).
However, in the mitochondria of other eukaryotes such as
yeast, RNase P does contain RNA (Miller and Martin,
1983). In the archaeon, Nanoarchaeum equitans, the RNase
P gene is missing, and no related catalytic activity is present
in cell lysates. Instead, in this archaeon, tRNAs are transcribed from position 1 of the tRNA genes, that is, they are
synthesised as leaderless tRNAs (Randau et al., 2008).
All tRNAs have a 3-terminal CCA sequence (positions

7476 in the standard cloverleaf representation). The CCA
sequence, which is universally conserved, is required for the
attachment of amino acids by aminoacyl-tRNA synthetases, as well as for interaction with ribosome during protein synthesis. This sequence is thought to control the
concentrations of active tRNA, adjusting them to match
translational activities. The ability to form the CCA
sequence is observed in all three domains of life (Yue et al.,
1996). In all eukaryotes, many archaea, and some eubacteria, some or all of the tRNA genes do not encode the 3terminal CCA sequence; instead, the sequence is added to
the premature transcript by ATP(CTP):tRNA nucleotidyltransferase (CCA-adding enzyme; Deutscher, 1990).
The CCA-adding enzyme is essential in organisms in which
not all tRNA genes encode CCA. Even in some eubacteria
that encode CCA in all the tRNA genes, such as E. coli, the
CCA-adding enzyme is nonetheless present. In these
organisms, the CCA-adding enzyme repairs exonucleolytically attacked tRNAs. Therefore, the CCA-adding
enzyme plays an important role in maintaining the content
of active tRNAs for use in translation. CCA-adding
enzyme is unique in that it synthesises a specic sequence
(CCA) onto specic premature tRNAs without the help of
a nucleic acid template. CCA-adding enzymes have been
categorised into two classes, I and II (Yue et al., 1996).
Archaeal CCA-adding enzyme belongs to class I, whereas
the eubacterial and eukaryotic enzymes belong to class II.
Although both classes of enzymes catalyse the same reaction, the two classes of enzymes do not share signicant
similarity at the primary amino acid sequence level. Class I
enzyme adopts a U-shaped structure, whereas class II
adopts a seahorse structure (Li et al., 2002; Okabe et al.,
2003; Xiong et al., 2003). Extensive structural and biochemical studies have resolved the detailed reaction
mechanism of these enzymes (Xiong and Steitz, 2004;
Tomita et al., 2004, 2006), but the mechanism of CCA
addition without a nucleic acid template remains
unknown. Compounding the mystery, in some slowly
evolved and deeply rooted eubacteria, CCA-adding activity is the collaborative eort of two distinct but closely
related CC- and A-adding enzymes (Tomita and Weiner,
2001, 2002). The structure of A-adding enzymes is similar
to that of CCA-adding enzymes. However, the molecular
basis for the diering specicities of class II enzymes
remains obscure.
In eukaryotic cells, 5 cleavage by RNase P and 3endonucleolytic cleavage are regulated by La protein,
which binds to precursor tRNA (Yoo and Wolin, 1997;
Intine et al., 2000). An interesting case has been identied
in the tRNA splicing of schyzon (Cyanidioschyzon merolae)
(Soma et al., 2007). That species possesses 11 tRNA genes
in which the 3 half of the tRNA lies upstream of the 5 half
in the genome (permutated tRNA genes). These genes are
expressed, and they produce mature tRNAs that are aminoacylated. The termini of the tRNA precursor are ligated,
resulting in formation of a characteristic circular RNA
intermediate that is then processed at the acceptor stem to
Table 2 tRNA splicing endonucleases, RNA ligases and references

tRNA splicing
endonuclease
RNA ligase
References
Eukaryotes
Yeast, fungi, plant
Heterotetramer
Multifunctional-type
Animalb
Heterotetramer
RtcB (Human
HSPC117)
Archaeac
a2, a4, a2b2 or e2
RtcB
(Trotta et al., 1997; Calvin and Li, 2008;

Hofmann et al., 2000; Schwer et al., 2008;
Phizicky et al., 1986; Wang and Shuman,
2005; McCraith and Phizicky,1991; Englert
and Beier, 2005)
(Englert et al., 2010; Filipowicz and
Shatkin,1983; Englert et al., 2011; Popow
et al., 2011)
(Englert et al., 2011; Marck and
Grosjean,2003; Tocchini-Valentini et al.,
2005; Fujishima et al., 2011; Li et al., 1998;
Randau et al., 2005; Xue et al., 2006; Trotta
et al., 2006; Yoshinari et al., 2009; Mitchell
et al., 2009; Hirata et al., 2011; Hirata et al.,
2012)
(Tanaka and Shuman, 2011; Reinhold-Hurek
and Shub, 1992)
Domain of life
a
Eubacteriad
a
Introns are found only between positions 38 and 39 (canonical position) in precursor tRNA. The secondary structure of cleavage sites forms the
buldgehelixbuldge (standard) motif.
Branchiostoma oridae (amphioxus), an exception among animals, has a multifuncitonal tRNA ligase.
c
Introns are often inserted into non-canonical positions such as D-arm, T-arm and aminoacyl stem in addition to the canonical position. The
secondary structures of cleavage sites in archaeal precursor tRNAs show several variations. Archaeal tRNA splicing endonucleases are classied
into four types (a2, a4, a2b2 and e2) based on the structures. The a2b2 and e2 types have a broad substrate specicity, in which nonstandard introns at
non-canonical positions are cleaved.
d
Some precursor tRNAs from proteobacteria have an intron. The intron is removed and the exons are joined by the group I intron self-splicing
mechanism. Consequently, specic endonucleases and RNA ligases are not required. RtcB homologues are widely found in eubacteria.
b
generate the correct termini after CCA addition. See also:

Transfer RNA Structure
tRNA splicing
Introns are often found in the tRNA genes of eubacteria,
archaea, eukaryotes and organelles. Most introns are
found between positions 38 and 39 in the anticodon loop
region. Table 2 summarises the enzymes concerned with
tRNA splicing; to minimise the citations in the text, the
table also provides detailed references. The mechanism of
tRNA splicing in yeast has been extensively studied by
identication of splicing mutants and purication of the
enzymes responsible for each step. In yeast, tRNA splicing
occurrs in the cytoplasm (Takano et al., 2005), and precursor tRNA is matured over the course of repeated
transports between the nucleus and cytoplasm (Ohira and
Suzuki, 2011). Yeast tRNA splicing endonuclease (a
complex consisting of Sen2, Sen15, Sen34, and Sen54)
cleaves intron-containing tRNA precursor at the canonical
splice site. The remaining 2,3-cyclic phosphate at the 3
end of the 5 fragment is converted to 2 phosphate by cyclic
phosphodiesterase. Subsequently, polynucleotide kinase
phosphorylates the 5 hydroxyl of the 3 fragment, and
adenylated tRNA ligase transfers its adenosine
8
monophosphate (AMP) to the 5 phosphate of the 3

fragment. Finally, the two RNA fragments are joined by
tRNA ligase, releasing AMP from the 5-terminus of the 3
fragment. In the nal step of the splicing process, the
remaining 2 phosphate of the 5 exon of the splice junction
is removed by 2 phosphotransferase. Plant and fungus
ligases have multiple functions, containing a polynucleotide kinase and cyclic phosphodiesterase domains as well as
a ligase domain. Thus, some steps of tRNA splicing in
plant, and fungi are catalysed by single proteins. In addition, Branchiostoma oridae (amphioxus) has a similar
multifunctional RNA ligase. In animals, 2,3-cyclic phosphate-dependent RNA ligase activity was initially detected
in HeLa cell extract. Recently, the corresponding RNA
ligase was identied as RtcB (HSPC117 in human). Similar
RNA ligases are present in many archaea and eubacteria.
In the case of archaea, introns are often inserted at noncanonical positions such as the D-arm, T-arm, and aminoacyl stem. Furthermore, the secondary structures of
cleavage sites in archaeal precursor tRNAs exhibit several
variations, whereas eukaryotic introns are found only in
the bulgehelixbulge motif at the canonical position.
Archaeal tRNA splicing endonucleases are classied into
four types (a2, a4, a2b2 and e2) based on their structures: the
a2b2 and e2 types have broad substrate specicities and can
cleave nonstandard introns at non-canonical positions. As

mentioned above, ligation in archaea is performed by the
RtcB homologue.
In proteobacteria, some precursor tRNAs contain
introns that are removed (and the exons joined) by the
group I intron self-splicing mechanism. Consequently,
specic endonucleases and RNA ligases are not required
for maturation of these tRNAs. See also: Transfer RNA
tRNA modification
Modied nucleotides have been found in many types of
RNA molecules, but tRNA molecules contain the greatest
diversity of such modications. At specic positions in
tRNAs, the normal nucleotides, A, G, U, and C, are
enzymatically converted to modied forms before, during,
or after the processing, splicing, and transport steps. To
date, more than 100 nucleotide derivatives have been
identied in tRNAs (Figure 2 and Figure 3). Although several
modications play important roles in the translation process (Table 2), the functions of many modications remain
to be discovered. To minimise the citation in the texts,
detailed references are provided in Table 1.
Modification at the wobble position

A number of modied nucleotides involved in codon
anticodon interactions are located at the rst position of
the anticodon (position 34 in the standard cloverleaf
representation in Figure 3). A single tRNA is often able to
decode multiple codons by forming a non-canonical base
pair (the wobble base pair) between the nucleotide at the
codons third position and nucleotide at the rst position of
the anticodon (the wobble position). Modications at the
wobble position can promote, expand, restrict and/or
alter codonanticodon interactions. Typical examples
include queuosine (Q), inosine (I), 5-formylcytidine (f5C),
lysidine (L, k2C), agmatidine (agm2C), and methylated
nucleosides (see Table 1). Uridine at the wobble position is
also modied to form various derivatives (xm5U or xmo5U),
such as 5-methylaminometyl-2-thiouridine (mnm5s2U),
uridine 5-oxyacetic acid (cmo5U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-taurinometyhl-uridine (tm5U) and
5-taurinometyl-2-thiouridine (tm5s2U). Although their
functions vary depending on the organism, in general these
uridine derivatives are involved in expanding or restricting
the decoding capacity of tRNA. Taurine-derived modied
nucleosides (tm5U and tm5s2U) have been found in animal
mitochondrial tRNAs. One intriguing nding is that the
tm5U-lacking tRNALeu(UUR) (anticodon, UAA) found in
cells of a human mitochondrial disease (MELAS: mitochondrial myopathy, encephalopathy, lactic acidosis and
stroke-like episodes) is able to decode UUA, but not UUG.
The UUG codon is mostly found in ND6 as a subunit of the
respiratory chain complex I, and loss of function of ND6 is
the primary defect in MELAS mutants. This result explains
the molecular mechanism by which the lack of a modied
nucleotide in tRNA can cause MELAS. In eubacteria, the

biosynthesis of xm5U modications requires multiple
enzymes
(Moukadiri et al., (2014)). However, the corresponding
biosynthetic pathways in eukaryotes (Chen et al., 2011) and
archaea (Tomikawa et al., 2013) have not been completely
claried. In the case of eukaryotes, Trm9 and the so-called
Elongator complex are involved. Trm9-dependent modications in yeast promote codon-specic translational
elongation and elevated levels of DNA damage response
proteins (Begley et al., 2007), which in turn inuence cellcycle regulation. The 2-thiolation of modied uridine and 2O-methylation of pyrimidine at the wobble position stabilise
the 3-endo form of ribose, and this conformational rigidity
can restrict or promote codonanticodon base pairing.
Modifications in the anticodon arm

Modied nucleotides are frequently found at the position 3adjacent to the anticodon (position 37). Because the
nucleotide at position 37 is conserved in all tRNAs as a
purine, all modied nucleotides at this position are derived
from G or A. In the case of G, the base is initially modied to
1-methylguanine (m1G), whereas A is either modied to one
of several derivatives or remains unmodied. The primary
function of modications at position 37 is the prevention of
frameshift errors. In addition to playing a role in readingframe maintenance, the m1G37 modication prevents mischarging of tRNAAsp by arginyl-tRNA synthase. In some
cases, modied nucleotides at position 37 are hypermodied
(wybutosine (yW; imG), N6-isopentenyladenosine (i6A), 2methylthio-N6-isopentenyladenosine (ms2i6A) N6-threonylcarbamoyladenosine (t6A), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) or their derivatives), and
they are involved in stabilizing codonanticodon interactions or the U-turn structure in the anticodon loop by
reinforcing stacking. Most tRNAPhe species of eukaryotic
origin, except for those of insects, contain wybutosine (yW;
imG) or its derivatives. The yW is synthesised from G via an
m1G precursor by the TYW1, -2, -3, and -4 enzymes. In
eubacteria, tRNAs that translate codons starting with U
contain i6A37, ms2i6A37, or their derivatives. Isopentenylpyrophosphate is utilised as an isopentenyl donor
by tRNA (i6A37) synthetase, encoded by miaA. t6A37, and
ms2t6A37 are present in tRNAs corresponding to codons
starting with A. For the synthesis of t6A, threonine is substrates for the modication reaction. Modied nucleosides
at positions other than 37 include 2-O-methylnucleosides,
5-methylcytydines, 2-methylguanosine, and pseudouridines
in the anticodon arm, which might aect anticodon-arm
structure. 2-O-methylation of ribose in the anticodon arm is
observed at positions 32, 34, and 39. The modication systems that perform these methylations dier among organisms. In some archaea, the modication is generated by
aFib, Nop5, and L7Ae in conjunction with the box C/D
guide RNA system. TruA (Pus1p in yeast) is a multiple sitespecic modication enzyme that specically modies U38,
NH
HN
CH3
HN
O
HO
HO
HO
HO
OH
HO
5
Pseudouridine ()
OH
HO
NH
HN
5-Methyluridine (m U)
or
ribothymidine (T)
OH
Dihydrouridine (D)
HO
HO
NH
O
N
HN
HO
H2N
HO
HO
HO
OH
HO
OH
HO
Inosine (I)
CH2NHCH2COOH
HN
HN
CH2
OH
Carboxymethylaminomethyl-2thiouridine (cmnm5s2 U)
Queuosine (Q)
CH3OOCCHNHCOOCH3
CH2
CH2
OCH2CO2H
HN
HNCH2CH
CH3
N
CH 3
CH 3
N
CH3
HO
HO
HO
OH
HO
Uridine 5-oxyacetic acid

(cmo5U)
HNCH2CH
N
CH3S
CH3
HO
2-Methylthio-N 6 2
isopentenyladenosine (ms i 6 A)
Figure 2
Modified nucleotides frequently found in tRNAs.
N 6 -Isopentenyladenosine (i6A)
CH3
CH3
CHOH
CHOH
HNCONHCHCO2H
HO
OH
OH
HNCONHCHCO2H
CH3
HO
OH
Wybutosine (yW)
HO
HO
CH3S
OH
N 6-Threonylcarbamoyladenosine
(t6A)
HO
HO
HO
OH
2-Methylthio-N 6threonylcarbamoyladenosine
(ms 2 t6 A)
74
73
72
71
1
2
70
69
68
A2-O-ribosyl phosphate
67
66
m1A
60 59
65 64 63 62 61
58
3
4
5
m2G,m22G
6
7
s4U
Arc haeos ine
8
D
16
17
18
19
Gm
15 14 13 12 11 10
20 21
m2G ,m2
2G
57
56
m5C
22 23 24 25 26
27
m2G ,m22G
75 76
49 50 51 52 53
54 55
48 47
28
29
43 44 45
42
41
30
31
40
39
32
33
34
38
37
35 36
46
T, s2T
m7G
m5C
m1G , yW , i6A, ms2i6A, t6A, ms2t6A, cyclic t6A
I, Q, f5C ,L , cmnm5s2U, c mo5U, mnm5s2U, m5U, m5s2U, m5C, agm2C

Figure 3 Location of modified nucleosides in tRNA. Numbering of nucleotides confirms to the secondary structure of yeast tRNAPhe; the solid and dotted
lines show the secondary and tertiary base pairs, respectively.
U39 and U40 to C38, C39 and C40, respectively, in the

anticondon arm.
Modifications in the three-dimensional core

Besides the anticodon arm region, modied nucleotides are
also found in the three-dimensional core of tRNAs. In
many cases, modied nucleotides in the core form tertiary
base pairs and stabilise the L-shaped structure of the tRNA
molecule (Figure 3). The main modied nucleosides in the
core are 4-thiouridine (s4U8), N2, N2-dimethylguanosine
(m22G) and its intermediate form 2-methylguanosine(m2G) at positions 6, 7, 9, 10, 18, 26, and 27; dihydrouridine (D16, 17, and 20); archaeosine (position 15); 2O-methylguanosine (Gm18); 7-methylguanosine (m7G46);
ribothymidine (T54); 2-thioribothymidine (s2T54); pseudouridine (C55); 1-methyladenosine (m1A57 and 58); and
A2-O-ribosyl phosphate at position 64. s4U8, which
engages in a tertiary interaction with A14, is formed by the
thiamine pathway enzymes ThiI and the cysteine desulphurase IscS. The IscS protein is also required for ecient
formation of s2T54 by the TtuA and TtuB proteins. In most
eukaryotic and eubacterial tRNAs, D is synthesised by the
dus gene product. Archeaosine, an archaea-specic modied nucleoside, is introduced at G15 in the D-loop by
archaeosine tRNA guanine-transglycosylase. In an E. coli
mutant lacking both Gm18 and C55, growth rate decreases

and the frequency of frameshift errors increases. Because
Gm18 engages in a tertiary interaction with C55, the Gm18
modication probably contributes to the formation of the
three-dimensional core. Gm18 methylation is catalysed by
tRNA (Gm18) methyltransferase (TrmH). A recent study
showed that the Gm18 modication in E. coli tRNA suppresses immunostimulation via Toll-like receptor 7. Thus,
enterobacteria exploit the Gm18 modication in tRNA to
avoid the host immune system. In the variable region, the
typical modied nucleoside m7G46 forms a tertiary base
pair with C13-G22 in the D stem. This modication is
catalysed by tRNA (m7G46) methyltransferase (TrmB in
eubacteria; Trm8-Trm82 complex in yeast). Thermophilic
TrmB enzymes have an extra C-terminal region, which
contributes to the protein stability and methylation
delity. T54 and C55 are frequently observed in the
T-loop. Two types of tRNA (m5U54) methyltransferase
are known: the AdoMet-dependent enzyme (TrmA) and 5,
10-methylenetetrahydrofolic acid-dependent enzyme
(TrmFO). T54, which engages in a tertiary interaction with
m1A58, stabilises the hydrogen bonds between the D- and
T-loops (C55-Gm18 and C56-G19). s2T54 is present in
thermophilic eubacteria and archaea, replacing the usual
T54 in eubacteria and contributes the thermostability of
tRNA (Watanabe et al., 1976). A sulphur relay system for
11
s2T54 modication is involved in the synthesis of molybdenum cofactor and thiamine (Shigi et al., 2008). C55 is
universally conserved among the three domains of life. C is
synthesised by TruB or Pus4p protein. m1A58, generated
by TrmI, is essential in thermophilic bacteria for growth at
high temperatures. Archaeal TrmI modies both A57 and
A58 to m1A57 and m1A58, respectively. The 2-O-ribosyl
phosphate A64 modication functions in distinguishing
initiator from elongator tRNAMet. See also: Transfer
RNA Modication
Summary
Synthesis of tRNA molecules requires a combination of
processes. tRNA synthesis shares processing and splicing
mechanisms with maturation of rRNA and mRNA molecules, whereas CCA addition and modication are, in
general, peculiar to tRNA molecules. The combination of
these mechanisms diers from species to species. This
complexity indicates that tRNA gene expression has
adopted new fundamental processes throughout evolution. Relative amounts of tRNAs are regulated by several
factors: copy number of tRNA genes; transcriptional
activity, derived from promoter activity mediated by
transcriptional factors and specic factors (ppGpp and
pppGpp in prokaryotes and Maf1 in eukaryotes) and
regulated by the nutrient conditions; and tRNA degradation by various nucleases. The evolutionary signicance of
the diversity of tRNA synthesis processes has not been well
characterised and needs to be claried. A remarkable
characteristic of tRNA, distinguishing it from both mRNA
and rRNA, is the presence of a wide variety of modied
nucleotides, which are introduced by modication
enzymes during or after the processing, splicing, and
transport. Although several modications are known to
play important roles in translation, the functions of many
modications remain to be discovered. In addition, many
of the modication processes have yet to be analysed.
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Microbiology.
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