doi: 10.1093/femsre/fuv004
Review Article
REVIEW ARTICLE
ABSTRACT
Transfer RNA is an essential adapter molecule that is found across all three domains of life. The primary role of transfer
RNA resides in its critical involvement in the accurate translation of messenger RNA codons during protein synthesis and,
therefore, ultimately in the determination of cellular gene expression. This review aims to bring together the results of
intensive investigations into the synthesis, maturation, modification, aminoacylation, editing and recycling of bacterial
transfer RNAs. Codon recognition at the ribosome as well as the ever-increasing number of alternative roles for transfer
RNA outside of translation will be discussed in the specific context of bacterial cells.
Keywords: aminoacylation; codon; modification; protein synthesis; translation; tRNA
INTRODUCTION TO tRNA
Transfer ribonucleic acid (tRNA) is an essential component of
the cellular protein synthesis machinery that was found at the
and Abelson 1979; Soll
and
origin of life (Altman 1978; Soll
RajBhandary 1995). Primary research on tRNA can be dated back
to the early 1950s when Hoagland et al. first determined the presence of an enzyme in the pH 5 insoluble fraction of a rat liver extract that could activate amino acids in an ATP-dependent manner with the concurrent production of aminoacyl-adenylates
(Hoagland, Keller and Zamecnik 1956; Hoagland, Zamecnik and
Stephenson 1957; Hoagland et al. 1958). After this initial finding,
further work in the Zamecnik laboratory demonstrated that the
amino acid acceptor molecule in the extract was in fact tRNA (reviewed in RajBhandary and Kohrer 2006). The first sequence of
a tRNA molecule was made available in 1965 after extensive research in the yeast model system (Holley 1965; Holley et al. 1965).
Since then, enhancement in column chromatography and gel
electrophoresis methodologies has resulted in rapid progress in
both purification and sequence determination of individual cellular tRNAs (Cherayil and Bock 1965; Gillam et al. 1967; Holladay,
Pearson and Kelmers 1971; Pearson, Weiss and Kelmers 1971;
280
281
Figure 1. The typical life cycle of a bacterial transfer RNA. The numbering system corresponds to the specific sections of this review. Abbreviations include aminoacyltRNA synthetase (aaRS), inorganic pyrophosphate (PPi), amino acid (aa) and elongation factor-TU (EF-Tu).
aminoacylated tRNA molecule will be required to undergo additional maturation steps prior to participation in these pathways (Blanquet et al. 2003). This review will focus specifically on
bringing together the key findings that have enhanced our understanding of the processes involved in the production of mature functional bacterial tRNA molecules and their interaction
with the ribosome and other biosynthetic pathways.
Synthesis of tRNA
In bacteria, the genetic material encoding tRNA is most frequently found in clusters on the chromosome and the presence
of multiple gene copies for a single species is common. To date,
three different tRNA-encoding transcription units have been described in the literature encompassing those that just contain
tRNA genes, those that contain tRNA genes in the spacer regions of ribosomal RNA operons and tRNA operons that also
include specific protein-encoding genes such as elongation factor Tu (EF-Tu) (Toh et al. 2001). These genetic structures enable
RNA polymerase-mediated transcription of a tRNA from a single promoter into precursors that are typically longer than the
final mature molecule (Altman 1975). Whilst they contain the
information required for constitution of a functionally unique
cellular molecule, in structural terms the genes encoding for
transfer RNA are often small ranging from only 75 to 90 base
pairs in length. The overall nucleotide sequence varies significantly between each tRNA gene but there are always either
four or five inverted repeats that are responsible for formation
of the stem-loop elements found within each cloverleaf structure (Altman 1978; Fournier and Ozeki 1985). In bacteria, includ-
ing Escherichia coli, the tRNA gene often encodes the critical 3
CCA sequence. This removes the requirement seen in eukaryotic systems whereby this feature must be specifically added to
the molecule post-transcriptionally. Aside from all of these tertiary structure-encoding components, tRNA genes also contain
specific recognition elements that are necessary for recruitment
of enzymes involved in precursor maturation, protein synthesis
and, in some cases, other non-translational pathways.
In terms of the necessity of specific promoter elements, it has
been demonstrated that the upstream regions of both E. coli and
Mycobacterium capricolum tRNA transcription units contain the
consensus regions of typical bacterial promoters including the
35 and 10 elements. In addition, the spacing between these
elements is consistent with that found for other bacterial pro and RajBhandary
moters, ranging from 15 to 18 nucleotides (Soll
1995). A conserved domain consisting of a 7 base pair sequence
that is GC rich and associated with the stringent control response has been identified in the promoter region of all E. coli
tRNA genes (Ikemura and Dahlberg 1973a,b). When focusing on
termination of tRNA transcription, DNA sequence analysis has
determined that, in both E. coli and M. capricolum, there are rhoindependent terminator structures that consist of a dyad symmetrical sequence and T-residues downstream of most of the
and RajBhandary 1995). However, it is thought
tRNA genes (Soll
that transcription of some tRNA genes, such as tRNATyr in E. coli,
may require rho-dependent termination. Further research is required to determine the specific details of the transcriptional
initiation and termination processes that lead to the successful
generation of immature tRNA precursor molecules in different
bacteria.
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Determination of the X-ray crystal structures of some examples of RNase P has resulted in the conclusion that the bacterial versions of the enzyme can be classified into two groups
(Krasilnikov et al. 2003, 2004; Kazantsev et al. 2005; Torres-Larios
et al. 2005; Kazantsev and Pace 2006; Baird et al. 2007). Type A, or
ancestral type RNase P, is widely dispersed throughout many of
the phylogenetic branches of the bacterial kingdom. In contrast,
type B or Bacillus type RNase P is restricted to Gram-positive bacteria (Pace and Brown 1995; Haas and Brown 1998; Baird et al.
2007). Whilst there are differences that focus around certain peripheral elements of the structures of type A and B RNase P proteins, both groups have a common catalytic core and tertiary fold
(reviewed in detail in Kazantsev and Pace 2006; Grosjean 2009).
Within the catalytic core, there are a series of highly conserved
residues. These residues occur within five separate regions of
sequence conservation encoding parts of the overall structure
that are involved in helix formation or the establishment of critical helical contacts. Structural studies on the protein components of type A and B RNase P have demonstrated that they
share both structural and functional similarity. This is further
supported by in vitro and in vivo studies that demonstrate efficient functional complementation of the protein component of
a type A RNase P with that of a type B RNase P and vice versa
(Waugh and Pace 1990; Wegscheid, Condon and Hartmann 2006).
The protein components of RNase P are known to complete the
typical ribonucleoprotein structure by folding into an - sandwich with a highly conserved electrostatic surface (Niranjanakumari et al. 1998; Stams et al. 1998; Spitzfaden et al. 2000; Kazantsev et al. 2003). A combination of mutational and biochemical
data has indicated that the unusual left-handed -- crossover
formed by the protein component produces a positively charged
face that binds to the RNA component of the enzyme (Stams
et al. 1998). Whilst the protein component of bacterial RNase P is
not required for catalytic activity in vitro, its overall importance
has been linked to acceleration of the tRNA processing reaction.
This suggests that it has a role in stabilization of the RNA component and possibly the reaction intermediates in addition to a
specific sequence in the precursor RNA in the C5 component of
RNase P (Walker and Engelke 2006; Smith, Hsieh and Fierke 2007;
Guenther et al. 2013).
When the tRNA precursor molecule contains one tRNA sequence, RNase P processing may be the only requirement for
efficient maturation of the 5 end. If the tRNA transcript contains more than one tRNA sequence other endoribonucleases,
such as RNase E and RNase III, may be required to generate
smaller precursor molecules. These smaller precursors can then
be much more efficiently processed by RNase P to generate a
tRNA molecule with a mature 5 end. Subsequent processing by
exoribonucleases, including RNases II, BN, D, PH, PNPase and T,
then account for generation of the mature 3 end (Sakano and
Shimura 1975, 1978; Schedl, Roberts and Primakoff 1976). Recently, RNase P-mediated separation of some E. coli transcripts
including valV valW, leuQ leuP leuV and secG leuU has been
demonstrated (Mohanty and Kushner 2007, 2008). One particular study reports RNase P-mediated separation of three precursor transcripts in E. coli that encode for the seven valine
tRNA molecules found within the cell (Agrawal, Mohanty and
Kushner 2014). Interestingly, specific focus on the ValU and LysT
transcripts has highlighted a previously uncharacterized role for
RNase P in 3 end processing. Unusually in this case, RNase P processing proceeds in the 3 to 5 direction generating one tRNA
precursor at a time with activity being initiated by specific endonucleolytic removal of the Rho-independent transcription terminators (Agrawal, Mohanty and Kushner 2014).
283
at the 3 end of the transcript usually leave a stretch of extra residues that must be removed by exoribonucleases (Schedl,
Roberts and Primakoff 1976). Two types of intermediate tRNA
precursors have been identified in bacterial cells and they differ in terms of their requirements for exonucleolytic processing
at the 3 end. In the most common type of precursor, the CCA
end of the tRNA is already present and is followed by a series
of extra nucleotides. Final maturation of the 3 end of this precursor requires a nuclease that can trim right up to the critical
CCA end without disrupting it. This is supported by mutagenesis
studies that directly demonstrate that the CCA repair enzyme,
nucleotidyltransferase, is not involved in the maturation of this
type of precursor (Deutscher, Lin and Evans 1977). In the second
type of precursor, which is rare in bacterial cells but common in
eukaryotes, other residues substitute the CCA end. In this case,
3 processing requires a nuclease that will remove only the substituting residues leaving a product that can be repaired by nucleotidyltransferase such that it has a CCA terminus at the completion of maturation.
Originally, it was thought that RNase II, a well-known 3 exonuclease, was the sole enzyme involved in 3 exonucleolytic
processing of tRNA transcripts. Whilst this enzyme has been
shown in vitro to remove extra nucleotides at the 3 end of an E.
coli tRNATyr precursor, the fact that it is processive in nature and
cannot stop at the CCA end has led to searches for another more
specific exonuclease (Cudny and Deutscher 1980). A more likely
candidate was found with the discovery of a monomeric 38 kDa
protein termed RNase D. RNase D is specific for tRNA precursors
that have the 3 CCA end. This enzyme specifically removes all
extra residues that come before the CCA end in a non-processive
manner and, in doing so, generates a mature precursor tRNA
molecule that has amino acid accepting capability (Cudny and
Deutscher 1980). Perhaps more importantly, RNase D cannot hydrolyze the CCA end and the rate of its hydrolytic activity has
been shown to reduce by 30-fold when it reaches this sequence
(Ghosh and Deutscher 1978; Cudny and Deutscher 1980).
In the case of the second type of tRNA precursor, which has
an incomplete or absent CCA end, another 3 processing exonuclease was identified termed RNase BN. RNase BN has a molecular weight of approximately 35 kDa and, unlike RNase D, its
specificity for tRNA precursors is not determined by the presence of an intact 3 CCA end (Seidman et al. 1975; Schmidt and
McClain 1978; Asha et al. 1983; Asha and Deutscher 1983). Other
exonucleases that have been shown to play a role in 3 tRNA processing include RNases PH, T and PNPase (described in some de and RajBhandary 1995). Studies attempting to fully
tail in Soll
characterize all of the separate components of the processing
machinery have shown that the role and order of activity of the
different enzymes is entirely dependent on the specific tRNA
precursor in question (Li and Deutscher 1996). The ultimate conclusion of all of the processes discussed in this section is the
generation of a mature tRNA molecule, which can then successfully progress into the nucleotide modification and aminoacylation pathways.
284
Modification
Enzyme
8
13
16
17
18
20
20a
32
S4 U
D
D
Gm
D
D
S2 C Cm, Um
IscS, Thil
TruD
Dus A, B, C
Dus A, B, C
TrmH
Dus A, B, C
Dus A, B, C
IscS, TtcA Trml RluA
34
I k2 C ac4 C Um, Cm Q,
GluQ mnm5 U cmnm5 Um
cmnm5 U mnm5 s2 U
cmnm5 s2 U mnm5 se2 U
mcmo5 U cmo5 U
37
m1 G m6 A m2 A i6 A ms2 i6 A
t 6 A m6 t 6 A
38
39
40
46
47
54
55
65
67
m7 G
acp3 U
rT
TruA
TruA
TruA
TrmB
?
TrmA
TruB
TruC
TruC
285
Figure 2. Known modifications and their positions in E. coli tRNA. The information presented in this figure was adapted from El Yacoubi, Bailly and de Crecy-Lagard,
(2012). Abbreviations for specific groups include m-, c-, n-, o-, t-, i-, K-, r- and s- for methyl, carbon, amino, oxy, threonine, isopentyl, lysine, ribose and thio groups,
respectively. Other abbreviations include acp: 3-amino-3-carboxypropyl, D: dihydrouridine, I: inosine, : pseudouridine, Q: queuosine. Other capital letters refer to
the appropriate bases uracil, cytosine, guanine and adenine. An index and an exponent indicate the number and the position of the substitution, for example 6dimethyladenosine is written as m6 2 A.
be a mechanism employed by pathogenic bacteria in host immune invasion (Gehrig et al. 2012). Further studies are required
to determine the nature and quantity of modifications involved
in this process.
Whilst tRNA modifications have a primary role in maintaining the efficiency and fidelity of translation, their importance is
generally underappreciated, and it is thought that they may also
take on a much more global regulatory role within the cell by
providing connections between protein synthesis, metabolism
and stress response pathways. This is reinforced by the finding
that many tRNA modifications are directly determined by cellular or environmental factors such as growth rate, oxygen levels
and nutrient levels. For example, the synthesis of some proteins
might be influenced by the proportion of tRNAs within the cell
pool that have a particular modification. This is because some
tRNA modifications give a bias for recognition of one codon over
another. In the case of the DNA damage response in yeast, tRNA
methyltransferase 9 has been found to enhance the translation
of DNA damage proteins, whose transcripts are overrepresented
by specific glutamic acid and arginine codons, by modifying the
Aminoacylation
The generation of aminoacylated tRNA is a critical function that
is essential for translation as well as a variety of other cellular
processes, which will be discussed in the section Other roles of
tRNA in the cell of this review. Across all three domains of life,
formation of an aminoacyl-tRNA is enzymatically a two-stage
process that is catalyzed by one of 23 aaRS. In the first stage of
286
Figure 3. Formation of an aminoacylated-tRNA molecule by an aaRS. An amino acid (blue) is activated by an aaRS to form an aminoacyl-adenylate. This process
requires ATP and results in the release of PPi. Following tRNA binding to the aaRS, the activated amino acid is transferred to the 3 end of the tRNA molecule forming
aminoacyl-tRNA with concurrent release of adenosine monophosphate (AMP). The aminoacyl-tRNA product is released from the aaRS and is either subject to binding
by EF-Tu for delivery to the ribosome or hijacking by other factors for diversion into biosynthetic pathways outside of translation. Figure reproduced from Reynolds,
Lazazzera and Ibba (2010).
and positions G1, G2, C81 and A82, which have been shown
to confer the amino acid accepting specificity of E. coli tRNATyr
(Hooper, Russell and Smith 1972; Shimura et al. 1972; Smith and
Celis 1973). The importance and relevance of specific tRNA acceptor stem elements can also vary between bacterial genera
and species. For example, it has recently been shown that Streptococcus pneumoniae AlaRS enzyme is less dependent upon the
G3:U70 acceptor stem wobble region for aminoacylation than
other bacterial AlaRS proteins (Shepherd and Ibba 2013b, 2014).
In addition, the same acceptor stem element can act as an antideterminant for aminoacylation by other aaRSs. For example, if
the G3:U70 element is put into yeast tRNAThr , then it becomes
an anti-determinant for aminoacylation by threonyl-tRNA synthetase (Nameki 1995; a full and comprehensive review of tRNA
identity elements is given in Giege, Sissler and Florentz 1998).
D-loop residues within tRNA can also act as aminoacylation
recognition elements for the aaRSs. This was first demonstrated
to be the case for yeast phenylalanyl-tRNA synthetase (PheRS),
which can aminoacylate heterologous tRNAPhe species that have
a specific sequence of bases in the D-loop that match that of its
own tRNA (Dudock et al. 1971; Roe, Sirover and Dudock 1973).
Strikingly, this region of tRNA structure has also been implicated
in editing of misaminoacylated-tRNA species by certain aaRSs
(see the Section Editing of aminoacyl-tRNA below for more details on the editing process). For example, D-loop residues 16,
20 and 21 in tRNAIle are implicated in the editing of mischarged
Val-tRNAIle by isoleucyl-tRNA synthetase (IleRS) (Hale et al. 1997;
Farrow, Nordin and Schimmel 1999). In addition, D-loop residue
16 in St. pneumoniae tRNAIle was recently shown to determine
the aminoacylation capacity of St. pneumoniae IleRS towards both
isoleucine and leucine (Shepherd and Ibba 2014).
Whilst the anticodon loop typically comes into close contact
with the aaRS in many systems (Schimmel et al. 1972; Schoemaker and Schimmel 1974; Schimmel 1977), its importance as a
specific identity element is dependent upon the particular tRNA
species. For example, early studies in yeast demonstrated that
the entire anticodon loop can be removed from tRNAPhe while
retaining the capacity for aminoacylation by PheRS (Thiebe,
Harbers and Zachau 1972). In contrast, mutation of the third
base in the anticodon of E. coli tRNAGly causes a significant decrease in the rate of aminoacylation of this species by glycyltRNA synthetase (Carbon and Squires 1971). In some instances,
the anticodon loop also confers the amino acid identity of the
species. If the second base in E. coli tRNATrp is mutated, then it
is readily misaminoacylated with glutamine (Yaniv et al. 1974).
In addition, modification of either of the first two bases in the
anticodon loop of E. coli tRNAfmet inactivates the species entirely
(Schulman and Pelka 1977).
After completion of aminoacylation, most charged tRNA
species are sequestered by EF-Tu and subsequently proceed
into protein synthesis at the ribosome. Alternatively they can
enter other specific cellular processes, as discussed below in
the Section Other roles of tRNA in the cell. However, some
aminoacylated-tRNA species need to undergo further maturation events prior to participation in these terminal processes as
described below.
Maturation of aminoacyl-tRNA
After aminoacylation, most tRNA species are ready to directly participate in protein synthesis at the ribosome or in
other cellular pathways. In a relatively small number of cases,
aminoacylated-tRNA must first undergo a further maturation
step. The most common examples of additional maturation
events that take place in bacteria include transamidation,
which generates both asparaginylated and glutaminylated tRNA
species, production of selenocysteinyl-tRNA and formylation of
initiator methionyl-tRNA (summarized in Fig. 4) (Blanquet et al.
2003).
Transamidation is employed by some bacterial species as an
indirect mechanism for producing correctly aminoacylated GlntRNAGln from Glu-tRNAGln and Asn-tRNAAsn from Asp-tRNAAsn
in the absence of an encoded glutaminyl- or asparaginyl-tRNA
synthetase, respectively. Transamidation was first identified as a
major pathway for generating Gln-tRNAGln in Gram-positive bac-
287
teria, cyanobacteria and certain organelles (Wilcox and Nirenberg 1968; Wilcox 1969; Schon, Hottinger and Soll 1988a; Schon
et al. 1988b). Further studies have since indicated that this pathway, as well as that for Asn-tRNAAsn production, can also be
found in some Gram-negative bacteria and archaea (White and
Bayley 1972; Strauch, Zalkin and Aronson 1988; Curnow, Ibba and
Soll 1996; Gagnon et al. 1996; Becker and Kern 1998; Tumbula
et al. 2000) (reviewed in Blanquet et al. 2003). Regardless of bacterial origin, the transamidation reaction can be divided into two
steps. Initially, tRNAAsn and tRNAGln are aminoacylated with aspartate and glutamate, respectively. This activity requires either
an aspartyl- or a glutamyl-tRNA synthetase that has very poor
or no discriminatory power against the non-cognate tRNA but
maintains specificity for the cognate amino acid. The second
stage of the reaction is then accomplished by an amidotransferase that uses either L-asparagine, in the case of tRNAAsn , or Lglutamate, in the case of tRNAGln , as an amide nitrogen donor to
generate the final products (Fig. 4A). The aminoacylated tRNA intermediates produced during the first stage of the reaction typically bind EF-Tu very poorly and so are not efficiently recruited
to the ribosome before the next stage of the reaction, which requires the amidotransferase, can proceed. In the case of AsntRNAAsn production, it has also been shown that binding of EFTu to the mischarged intermediate is prevented by formation of
a protective shuttling complex, called the transamidosome, between the non-discriminatory AspRS and the amidotransferase
(Bailly et al. 2007).
Characterization of the glutamate amidotransferase (GluAdT) from Bacillus subtilis has demonstrated that the protein
is produced by expression from three genes, termed gatC, gatA
and gatB to produce a final product that is heterotrimeric
in nature (Strauch, Zalkin and Aronson 1988; Curnow et al.
1997). Since B. subtilis does not have a Gln-tRNA synthetase,
Figure 4. Indirect tRNA aminoacylation pathways. (A) The indirect pathways for generation of Gln-tRNAGln and Asn-tRNAAsn by transamidation. Abbreviations include
AdT for amidotransferase. (B) The selenocysteine tRNA modification pathway. (C) The formylation pathway for initiator Met-tRNAfMet . Abbreviations include FMT for
methionyl-tRNAf Met transformylase, FTHF for N-10 formyltetrahydrofolate and IF2 for initiation factor 2.
288
transamidation in the presence of a non-discriminatory GlutRNA synthetase is the only mechanism for producing correctly
acylated Gln-tRNAGln in this bacterium, thus indicating the importance of specific aminoacyl-tRNA maturation events for cell
survival (Lapointe, Duplain and Proulx 1986; Schon, Hottinger
and Soll 1988a; Chen et al. 1990). In many cases, including
Deinococcus radidurans, Thermus thermophilus and B. subtilis, GluAdt has been shown to have dual activity in vitro (Curnow et al.
1997, 1998; Becker et al. 2000b). Therefore, the in vivo specificity of
this enzyme against Asp-tRNAAsn or Glu-tRNAGln is entirely dependent on the cellular context, particularly with regard to the
presence or absence of the necessary tRNA-synthetase enzymes
(Becker et al. 1997, 2000a; Curnow et al. 1998). Maturation of GlutRNAGln and Asp-tRNAAsn to the correctly acylated species is essential for maintaining the fidelity of the genetic code within the
bacteria that possess these pathways.
Another form of aminoacyl-tRNA maturation is described
by the selenocysteinyl-tRNA pathway, which is found across all
three domains of life (reviewed in Commans and Bock 1999). The
requirement for this maturation pathway has been found to be
most notable in the synthesis of oxidoreductases, which often
substitute selenocysteine at cysteine codons because of its lower
redox potential (Blanquet et al. 2003). The first detection of a protein containing selenocysteine was made in the mid-1970s in
a glycine reductase isolated from the anaerobic Gram-positive
bacterium, Clostridium sticklandii (Cone et al. 1976). Since then the
pathway has been well characterized in E. coli, where it was first
shown that co-translational insertion of selenocysteine occurs
at UGA codons that are present within a specialized mRNA context (Chambers et al. 1986; Zinoni et al. 1986).
Detailed characterization of the E. coli selenocysteinyl-tRNA
pathway has indicated that this bacterium has a specific gene,
called selC, which encodes the genetic material required for production of a unique tRNA molecule that is specific for selenocysteine and displays an anticodon that is complimentary to UGA
(Leinfelder et al. 1988). This tRNASec molecule is a substrate for
serylation by seryl-tRNA synthetase. After the production of SertRNASec , a 500 kDa selenocysteine synthase (SelA) modifies the
serine into selenocysteine using phosphoselenate (produced by
SelD) as a donor (Fig. 4B).
The efficiency of the SerRS:tRNASec mischarging reaction has
been demonstrated to be as low as 1% compared to that of the
five cognate tRNASer isoacceptors in E. coli (Amberg et al. 1996;
Commans and Bock 1999). The low rate of serylation of tRNASec
by SerRS can be explained by the unusual and unique structure
of the tRNA molecule. Bacterial tRNASec is known to be larger
than other tRNAs and, in E. coli, reaches a total of 95 nucleotides
in length. The increased size is due to a combination of features including a long extra arm that is required for recognition by SerRS, and an extended 13 base pair acceptor helix. In
addition, tRNASec has been shown to have a purine: pyrimidine
pair between positions 11 and 24, which is rare except in initiator tRNAs, an extended 6 base pair D-stem and a reduced 4
nucleotide D-loop (Baron et al. 1993; Sturchler et al. 1993; Commans and Bock 1999). These specific and unique tRNA features
have been demonstrated to contribute to various stages of the
decoding process (Commans and Bock 1999).
Decoding and distinction of selenocysteine codons from
normal UGA stop codons also requires the presence of a
stem-loop structure, called the SECIS element, to be located
approximately 40 nucleotides away from the 3 end of the UGA
selenocysteine codon. In addition, a specific elongation factor,
called SelB, is required for recognition of the SECIS element and,
thus, efficient delivery of mature selenocysteinyl-tRNASec to the
Editing of aminoacyl-tRNA
Under certain circumstances, non-cognate aminoacyl-tRNA
needs to be proofread and edited to ensure that the efficiency
and fidelity of translation is maintained. In bacteria, this is
achieved by a combination of enzymatic activities, which include the pre- and post-transfer editing mechanisms of aaRSs
as well as the functions of trans-editing factors, peptidyl-tRNA
hydrolase and D-aminoacyl-tRNA deacylase.
The aaRSs represent the first step in quality control of translation (reviewed extensively in Ling, Reynolds and Ibba 2009;
Reynolds, Lazazzera and Ibba 2010). Aminoacyl-tRNA synthesis
occurs in two stages (described in the Section Aminoacylation
and Fig. 3) and, if the accuracy of translation is to be adequately
maintained, the aaRSs must successfully sequester both cognate tRNA and amino acid from similar non-cognate molecules
present in the cellular pool. Generally, the aaRSs have high specificity for their cognate tRNA. This is due to a combination of
the large surface area that is available during recognition of this
substrate and also kinetic proof reading that occurs during the
aminoacylation reaction (Ibba and Soll 2000; Guth and Francklyn
2007). However, near-cognate amino acids can be much harder
for the aaRSs to discriminate against since they can differ from
the cognate substrate by as little as one methyl group. This particular problem has been investigated in detail for IleRS, which
must distinguish between two isosteric amino acids: isoleucine
and valine. In this case, IleRS is able to maintain the accuracy
of translation by employing both pre- and post-transfer editing
289
Figure 5. Quality control steps during the formation of a non-cognate aminoacyl-tRNA. After activation of a non-cognate amino acid (red), the aminoacyl adenylate
may be hydrolyzed and released. A non-cognate aminoacyl-tRNA may be translocated into the editing site of the aaRS for hydrolysis. If the mischarged aminoacyl-tRNA
is released from the aaRS without being edited, it is subjected to editing by trans-editing factors or through resampling by the aaRS. Some non-cognate aminoacyltRNAs are discriminated against by EF-Tu, which either binds them too tightly or too weakly for efficient delivery and release in the ribosome. Figure reproduced from
Reynolds, Lazazzera and Ibba (2010).
290
one and two bind the ribosome in a process that is aided by GTPactivated release factor three. This occurs only in response to a
stop codon arriving in the aminoacylation site (Weissbach 1977)
(reviewed fully in Ogle, Carter and Ramakrishnan 2003; Khade
and Joseph 2010; Novoa and Ribas de Pouplana 2012).
During each stage of translation, tRNA undergoes a series of
specific interactions with the ribosome that participate directly
in the decoding process. In the earliest stages of translation, interactions between the anticodon loop of initiator fMet-tRNAfmet
and the P site of the 30S ribosomal subunit are directly responsible for the high accuracy of initiation factor three-mediated discrimination against elongator tRNA. More specifically, residues
G1338 and A1339 within the 16S rRNA component of the small
subunit interact with conserved GC base pairs found at initiator tRNA anticodon arm positions 2941 and 3040, respectively.
This stabilizes docking of initiator tRNA to the ribosome and results in full initiation of translation (Abdi and Fredrick 2005; Lancaster and Noller 2005; Berk et al. 2006; Korostelev et al. 2006;
Selmer et al. 2006; Qin, Abdi and Fredrick 2007; Voorhees et al.
2009).
After completion of early initiation, the acceptor stem of the
aminoacylated-tRNA is bound in a complex with EF-Tu and so
cannot interact with the A site of the 50S ribosomal subunit.
However, the anticodon loop of the tRNA is able to directly interact with the A site of the 30S ribosomal subunit (Moazed and
Noller 1989). Conserved residues G530, A1492 and A1493 within
the 16S rRNA of the small subunit interact with the resulting
codon-anticodon helix (Ogle et al. 2001; Cochella, Brunelle and
Green 2007). Together, these interactions result in the formation
of a decoding intermediate, which is most commonly referred
to as the A/T state. This state is not stable during interaction between the ribosome and aminoacyl-tRNA that does not match
the codon specified by the bound mRNA sequence (Pape, Wintermeyer and Rodnina 1999; Valle et al. 2003).
Interaction of the aminoacylated-tRNA with the A site of the
50S ribosomal subunit is only achieved upon successful codon
anticodon pairing, which triggers EF-Tu-mediated GTP hydrolysis. Accommodation of the aminoacylated-tRNA within the 50S
A site subsequently triggers peptide bond formation by the 23S
rRNA that comprises the peptidyl-transferase center (Ban et al.
2000; Nissen et al. 2000; Harms et al. 2001). This reaction is known
to be aided by the formation of a base pair between G2553 of 23S
rRNA and residue C75 of the aminoacyl-tRNA present in the ribosomal A site (Schmeing et al. 2005). After this reaction, deacylated tRNA becomes accommodated in the P site of the ribosome, eventually shifting to the E site where the acceptor stem
undergoes various interactions with the 50S ribosomal subunit.
Dipeptidyl-tRNA in the A site can then progress to the P site
making room for accommodation of another aminoacyl-tRNA,
thus allowing translation to proceed (fully reviewed in Khade
and Joseph 2010). Aminoacylated-tRNA species that are not recruited by the translation machinery typically proceed into other
cellular pathways as described below.
291
bridge requires the sequential activities of the Gly-tRNAGly dependent FemX, FemA and FemB proteins (Rohrer et al. 1999;
Tschierske et al. 1999; Schneider et al. 2004). Three out of the five
tRNAGly isoacceptors encoded in the genome of Sta. aureus have
sequence identity elements consistent with weak binding to
EF-Tu, thus ensuring their participation in peptidoglycan rather
than protein synthesis (Giannouli et al. 2009). Participation of
aminoacyl-tRNA in Sta. aureus cell-wall biogenesis is critical, and
selective inactivation of femA or femB is known to be lethal due
to a combination of phenotypic effects that include loss of methicillin resistance (Stranden et al. 1997; Ling and Berger-Bachi
1998; Rohrer and Berger-Bachi 2003). Two other Staphylococcal species, Sta. simulans and Sta. capitis, use the aminoacyltRNA-dependent Lif and Epr proteins, respectively, to substitute serine at the third and fifth positions of the standard pentaglycine bridge. By doing so, these proteins ultimately confer resistance against the glycylglycine endopeptidases that are produced by these bacteria, thus ensuring that peptidoglycan integrity is maintained during competition with other species for
occupation of the same niche (Sugai et al. 1997; Tschierske et al.
1997; Ehlert et al. 2000). Other well-characterized examples of the
importance of aminoacylated tRNAs in peptidoglycan biosynthesis include Weissella viridescens FemX (Ala addition), Enterococcus faecalis BppA1/BppA2 (Ala addition) and the vancomycindependent FemX/VanK system (Gly addition) in Streptomyces
coelicolor (reviewed in Shepherd and Ibba 2013a) (Bouhss et al.
2002; Hegde and Blanchard 2003; Hong et al. 2004, 2005; Maillard
et al. 2005; Fonvielle et al. 2009).
In addition to their involvement in peptidoglycan biosynthesis, aminoacylated-tRNAs also participate in antibiotic resistance and synthesis pathways. Many Gram-positive and Gramnegative bacteria have a specific type of virulence factor, called
MprF, encoded in their genome that can catalyze the aminoacylation of inner membrane lipids subsequently altering the
permeability of the cell wall to cationic antimicrobial agents
(reviewed in Dare and Ibba 2012; Shepherd and Ibba 2013a).
This process requires donor aminoacyl-tRNA molecules and is
known to adjust the overall negative charge of the cell membrane in a manner that is dependent upon the nature of the
amino acid that is being transferred to the lipid (Roy and Ibba
2008; Ernst and Peschel 2011). One of the most well characterized classes of MprF protein is comprised of the aminoacylphosphatidylglycerol synthases (aaPGSs). The aaPGSs are integral cytoplasmic membrane proteins that specifically transfer
amino acids from appropriately charged tRNA species to the
polar head group of phosphatidylglycerol, which is a common
bacterial membrane lipid (Roy and Ibba 2008; Ernst et al. 2009).
Lysylation of phosphatidylglyercol by the aaPGS enzymes of C.
perfringens, Sta. aureus and M. tuberculosis is associated with an
increase in the net positive charge of the cytoplasmic membranes of these bacteria. This change in membrane polarity has
been directly correlated with reduced permeability to defensins,
aminoglycosides, glycopeptides and -lactams (Oku et al. 2004;
Staubitz et al. 2004; Roy and Ibba 2008; Maloney et al. 2009). In
contrast, recent work focusing on Listeria monocytogenes aaPGS
has indicated that lysylation of phosphatidylglycerol in the
membrane of this pathogen has a much more global physiological effect on the cell that extends beyond antimicrobial resistance and into regulation of the motility operon (Dare et al.
2014). In addition, Pseudomonas aeruginosa has an aaPGS enzyme
that aminoacylates phosphatidylglycerol with alanine resulting
in neutralization of the overall charge of the membrane. This
subsequently causes enhancement in the resistance of the bacterium to a range of negatively charged -lactams and increased
292
OUTLOOK
Recent years have seen new fields opening up in tRNA biology, the success of which is due in no small part to the substantial body of knowledge that has been built up over the last
50 years. Advances in genomics and other technologies that can
be applied to the entire cellular population of tRNAs have started
to reveal new roles for tRNAs as regulators of numerous cellular processes (Raina and Ibba 2014). For example, the ability to
measure the aminoacylation status of all tRNAs and to monitor tRNA modification patterns globally have begun to uncover
new roles for tRNA in cellular stress responses (Pan 2013; Gu,
Begley and Dedon 2014). Another area of growing interest comes
from the increasing awareness that tRNAs directly and indirectly
function in numerous roles outside of translation of the genetic
code (Banerjee et al. 2010; Anderson and Ivanov 2014). Overall,
research in the tRNA field is entering a new and exciting era and
there are clearly many more surprises still to come from this
small but highly versatile RNA.
FUNDING
This work was supported by a grant from the National Institutes
of Health (GM065183).
Conflict of interest. None declared.
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