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TRENDS in Biochemical Sciences
Vol.30 No.12 December 2005
Amino acid specificity in translation
Taraka Dale and Olke C. Uhlenbeck
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA
Recent structural and biochemical experiments indicate
that bacterial elongation factor Tu and the ribosomal
A-site show specificity for both the amino acid and the
tRNA portions of their aminoacyl-tRNA (aa-tRNA)
substrates. These data are inconsistent with the
traditional view that tRNAs are generic adaptors in
translation. We hypothesize that each tRNA sequence
has co-evolved with its cognate amino acid, such that all
aa-tRNAs are translated uniformly.
Introduction
The mechanism of protein synthesis is traditionally
considered to have two phases with different specificities
towards the 20 amino acid side chains (Figure 1). In the
first phase, each amino acid is specifically recognized by
its cognate aminoacyl-tRNA synthetase (aaRS) and
esterified to the appropriate tRNA to form an aminoacyltRNA (aa-tRNA). In the second phase, all of the different
aa-tRNAs are funneled into the translational machinery
by binding elongation factor Tu$GTP (EF-Tu$GTP; EF-1
in eukaryotes) to form a ternary complex, which subsequently binds to the ribosome. The traditional view has
been that the components of this second phase are not
specific for the type of amino acid, and that tRNAs are
generic adaptors that are entirely specified by the anticodon. As summarized by Woese [1], this view that tRNAs
are adaptors which connect the amino acid with the
anticodon was hypothesized on theoretical grounds by
Crick in 1958 [2] and immediately accepted as the
paradigm. This lack of specificity for the amino acid in
the second phase of translation ensures that all amino
acids are incorporated into protein with similar efficiencies and rates, despite their characteristic differences in
size, charge and hydrophobicity.
Experiments showing that misacylated tRNAs can be
incorporated into protein supported the paradigm by
suggesting that the translational machinery does not
recognize the esterified amino acid of aa-tRNA. The classic
Chapeville experiment using Raney Nickel to convert
Cys-tRNACys to Ala-tRNACys found that alanine was
incorporated at the cysteine codons in an in vitro translation assay [3]. This result demonstrated that the translational machinery is unable to distinguish an incorrect
from a correct amino acid. Many additional examples of
incorporation of amino acids from misacylated tRNAs into
protein have since been reported [4–6]. Perhaps the most
extensive experiments evaluating the incorporation of
misacylated tRNAs relied on measuring the extent of
suppression of nonsense codons by suppressor tRNAs.
Corresponding author: Uhlenbeck, O.C. ([email protected]).
Available online 2 November 2005
For example, in the course of deducing the recognition
rules of aaRSs, several amber-suppressor tRNA bodies
were deliberately mutated such that they were aminoacylated by a different aaRS, and the resulting ‘identityswapped’ tRNAs were shown to insert the new amino acid
into protein [7,8]. In addition, suppressor tRNAs esterified
with O30 different unnatural amino acids have been
successfully incorporated into protein [9]. Together, these
data suggest that the translational apparatus lacks
specificity for different amino acids, once they are
esterified onto tRNA.
In a few isolated cases, however, the translation
machinery seems to show specificity for the esterified
amino acid. A prominent example occurs in the transamidation pathway, which is used as an alternative to GlnRS
to produce Gln-tRNAGln in many bacteria and archaea
[10,11]. In this pathway, tRNAGln is first misacylated by
GluRS to form Glu-tRNAGln and then reacted with a
specific amidotransferase to produce Gln-tRNAGln.
Because organisms using this pathway do not show misincorporation of glutamic acid at glutamine codons, it
seems that the misacylated Glu-tRNAGln intermediate of
this pathway is not translated. However, in vitro experiments show that, although Gln-tRNAGln can bind EF-Tu,
Glu-tRNAGln binds poorly [12]. As a result, the amidotransferase can successfully compete with EF-Tu for the
misacylated Glu-tRNAGln. Thus, in this case, the translational machinery seems to discriminate against certain
misacylated tRNAs.
Hypothesis: amino acid specificity in translation
promotes tRNA diversity
Although the bacterial translation apparatus shows little
specificity for cognate aa-tRNAs, it does show specificity
for the esterified amino acid portion of each aa-tRNA.
In the case of EF-Tu, this specificity is only observed by a
quantitative analysis of the binding properties of misacylated tRNAs. In the case of the ribosome, the X-ray
structure and the majority of the biochemical data suggest
that the ribosomal A-site also shows specificity for binding
the esterified amino acid. We hypothesize that, for each
aa-tRNA to function equivalently in translation, each
tRNA sequence has evolved to adjust its affinity for EF-Tu
and for the ribosome in a way that compensates for the
particular affinity of its cognate amino acid. As a result,
tRNAs are not generic, interchangeable adaptors, but
have individually evolved to be translated uniformly.
Crystal structures of three amino-acid-binding pockets
Amino acid binding in translation can be pictured using
several available X-ray crystal structures. Figure 2
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TRENDS in Biochemical Sciences
Vol.30 No.12 December 2005
(a) Phase I: aa-tRNA synthesis
aaRS
PPi
Amino acid
ATP
AMP
aaRS
tRNA
(b) Phase II: translation
aa-tRNA
EF-Tu GTP
Ternary complex
formation
E
P
A
Ribosome with empty A-site
Ternary
complex binding
and decoding
E
P
EF-G GDP
+ Pi
A
Ribosome with
empty A-site
E
A
Translocation
GTP
hydrolysis
EF-G GTP
Peptidyl
transfer
E
P
P
A
Pi
Accommodation
E
P
A
E
P
A
EF-Tu GDP
Ti BS
Figure 1. The two phases of translation. (a) Phase I, the specific phase, involves the ATP-dependent aminoacylation of a given tRNA by its cognate aminoacyl-tRNA synthetase
(aaRS). An example reaction (red) of the formation of Gln-tRNAGln by GlnRS is shown. Although other tRNAs (other colors) have a similar overall shape, their different
nucleotide sequences and repertoire of post-transcriptional modifications preclude them from interacting with GlnRS, and only glutamine precisely fits into the amino-acidwww.sciencedirect.com
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TRENDS in Biochemical Sciences
compares the amino-acid-binding pockets of TyrRS [13],
EF-Tu [14] and the A-site of 50S ribosomes [15], each of
which was co-crystallized with an amino acid or amino
acid analog. In the case of TyrRS, the bound tyrosine
precisely fits into the pocket and makes numerous
hydrogen bonds and Van der Waals contacts with the
protein (Figure 2a). In the case of EF-Tu, which is
complexed with Phe-tRNAPhe, the esterified phenylalanine fits in a large crevice in the protein and seems to
stack with His67 (Figure 2b). In addition, several
hydrogen bonds form between the a-amino group of the
esterified phenylalanine and EF-Tu. Finally, the aminoacid-binding pocket in the ribosomal A-site differs from
the previous two in that it is composed entirely of rRNA. In
this case, the p-methoxyphenyl portion of the puromycin
binds in a cleft formed by two rRNA bases, presumably
deriving stability via stacking interactions (Figure 2c).
At first glance, the three amino-acid-binding pockets
seem to be well suited for their required functions. TyrRS,
like all aaRSs, uses its pocket to specifically select its
cognate amino acid from the 19 non-cognate amino acids,
which is crucial for properly pairing the amino acid and
tRNA during aa-tRNA formation. Smaller amino acids
cannot form all of the stabilizing hydrogen bonds, whereas
larger amino acids will not fit into the pocket. By contrast,
EF-Tu and the ribosomal A-site have pockets that are
large enough to fit any amino acid side chain, which is
consistent with their need to accept all of the different
aa-tRNAs as substrates. However, because the pockets of
both EF-Tu and the A-site seem to form stacking
interactions with aromatic amino acids, these amino
acids might bind with higher affinity than smaller amino
acids, which cannot stack. This raises several questions:
† How do other amino acid side chains fit into the EF-Tu
and A-site pockets?
† Do the residues that form the pockets retain a rigid
structure, or do they rearrange to accommodate the
different amino acids?
† What are the binding affinities of the different amino
acids for each pocket?
From the limited data, it seems that some repositioning
of pocket residues can take place. An X-ray crystal
structure of EF-Tu complexed with Cys-tRNACys shows
that small rearrangements of the amino-acid-binding
pocket occur to enable the sulfhydryl group to pack
against Asn285 instead of stacking with His67 [16].
However, the consequences of this rearrangement with
regard to amino acid specificity are unclear. Thus, the
crystal structures indicate that the amino-acid-binding
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661
pockets of EF-Tu and the ribosomal A-site could easily
show specificity for the different amino acid side chains.
The hidden specificities of EF-Tu
As would be expected for a protein that must bind multiple
substrates, EF-Tu binds to all cognate aa-tRNAs within a
narrow range of affinities [17,18]. This uniform binding is
consistent with the idea that EF-Tu is a non-specific
binding protein that does not discriminate between
different amino acids or tRNA sequences. However, other
experiments using misacylated tRNAs have revealed a
‘hidden’ specificity of EF-Tu for both the amino acid side
chain and the tRNA. For example, early studies showed
that Phe-tRNALys binds to EF-Tu approximately fivefold
tighter than the cognate Lys-tRNALys [19,20]. An expanded
study used four tRNAs and four amino acids to form the 16
possible combinations of aa-tRNAs [21]. As expected, the
four cognate aa-tRNAs bound EF-Tu with similar affinities; however, the binding affinities of the 12 misacylated
tRNAs to EF-Tu varied dramatically from 13 times weaker
to 400 times stronger than the cognate aa-tRNAs.
Subsequently, tRNAPhe was misacylated with 13 different
amino acids, and their affinities for EF-Tu were found to
vary from 1.9 nM to 150 nM (w80-fold) [22]. Furthermore,
19 different tRNA bodies misacylated with the same amino
acid (valine) and displayed a 700-fold range in affinities
(0.44–310 nM) [23]. Taken together, these data demonstrate that both the amino acid side chain and the tRNA
body make highly varied and independent thermodynamic
contributions to EF-Tu binding, and that these contributions compensate for one another such that the overall
binding of cognate aa-tRNAs is uniform (Figure 3). Thus,
the apparent lack of specificity of EF-Tu for cognate aatRNAs is actually the result of two specific, but opposing,
interactions between the protein and the aa-tRNA.
The experiments involving misacylated tRNAs and
EF-Tu demonstrate that, rather than being a generic
adaptor, each tRNA has an important role in ensuring
uniform binding of aa-tRNA to EF-Tu. Parts of the
sequence of each tRNA have evolved to compensate for
the variable contribution of its esterified amino acid to the
overall binding affinity. For example, to compensate for
the weak binding of alanine, tRNAAla contains sequence
elements that ensure that it binds to EF-Tu stronger than
tRNAGln, which is associated with the strongly binding
glutamine. Because the co-crystal structure of the ternary
complex shows that the protein primarily interacts with
the phosphodiester backbone of the acceptor and T stems
of tRNA [14,16], it is likely that sequence differences
binding pocket of GlnRS. For each aaRS, the aminoacylation reaction is highly specific for both the amino acid and the tRNA, such that the incorrect formation of a
misacylated tRNA is estimated to be one in 10 000 [11]. (b) In phase II, the non-specific phase of translation, all aa-tRNAs are funneled into a common translational apparatus
(grey). Each aa-tRNA forms a ternary complex with elongation factor Tu (EF-Tu) and GTP, and binds to the ribosome in process termed decoding. Correct codon–anticodon
pairing between the A-site mRNA codon and the tRNA anticodon during decoding activates the GTPase activity of EF-Tu [43,44], and GTP hydrolysis occurs. A subsequent
conformational change is induced in EF-Tu, which results in the release of aa-tRNA and enables the acceptor end of the aa-tRNA to move into the A-site in a process termed
accommodation. After accommodation, the growing polypeptide esterified to the P-site-bound tRNA is transferred to the A-site-bound tRNA, elongating the peptide chain by
one amino acid. With the aid of elongation factor G (EF-G), the deacylated P-site tRNA is then translocated to the E-site, and the A-site-bound tRNA is translocated to the P-site.
The ribosomal A-site is then available for binding to the next ternary complex. These steps were discerned from a series of kinetic measurements [27]. The structures of GlnRS
and the GlnRS–tRNAGln complex are from PDB file 1GSG [45]. The tRNA, EF-Tu$GTP, and ternary complex structures are from PDB file 1TTT [14]. EF-Tu$GDP structure is from
PDB file 1TUI [46]. The complex structure of the ribosome and detailed positioning of the aa-tRNAs on the ribosome were omitted for simplicity; molecules are not drawn to
scale.
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Opinion
TRENDS in Biochemical Sciences
Vol.30 No.12 December 2005
Figure 2. Amino-acid-binding pockets in translation. All three pockets are viewed from the solvent-accessible side. (a) X-ray crystal structure of the tyrosine-binding pocket of
Escherichia coli TyrRS (PDB code: 1X8X) [13]. The surface representation of the pocket (i) shows a precise fit between the bound tyrosine (green) and the pocket (colored by
electrostatic potential: red, negative; blue, positive). The stick representation (ii) reveals putative hydrogen bonds (broken lines) between the p-hydroxyl group of the bound
tyrosine and protein residues Tyr37 and Asp182, which aid in achieving substrate specificity. In addition, the a-amino group of the bound tyrosine is within hydrogenbonding distance of Gln179, Tyr175 and Asp81, which might further stabilize the position of the substrate. (b) X-ray crystal structure of the amino-acid-binding pocket on the
surface of Thermus aquaticus EF-Tu (PDB code: 1TTT) [14]. The 3 0 -terminal adenosine and the esterified phenylalanine of Phe-tRNAPhe are shown (green). The surface
representation (i) shows the esterified phenylalanine bound in a spacious pocket that seems to be large enough to accommodate all 20 amino acids. The stick representation
of the pocket (ii) shows that the a-amino group of the esterified amino acid can form hydrogen bonds with main-chain protein residues Asn285 and His273. These interactions
can be formed with all amino acids except proline. (c) X-ray crystal structure of puromycin (green) bound to the A-site of the 50S subunit of Haloarcula marismortui ribosomes
(PDB code: 1KQS) [15]. Puromycin is an antibiotic analogous to the 3 0 -terminal adenosine and esterified amino acid of an aa-tRNA. Surface (i) and stick (ii) representations
show that the large, aromatic p-methoxyphenyl side chain is in a large pocket formed on one side by the imperfectly stacked bases A2486 and C2487 (A2451 and C2452 in
E. coli). Aromatic amino acids should bind to this pocket better than small aliphatic groups [47].
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between tRNAGln and tRNAAla in this part of the molecule
are responsible for the differences in affinity. However, the
precise manner in which the sequence of an RNA duplex
affects the positioning of its phosphodiester backbone is
not completely understood; therefore, the rules for tRNAsequence specificity for EF-Tu remain to be established.
Does the ribosome display amino acid specificity?
Of the steps in the elongation cycle (Figure 1) that could be
sensitive to the identity of the amino acid, the accommodation step is the best candidate. This step, which might
consist of several sub-steps [24], involves the release of the
acceptor end of aa-tRNA from EF-Tu$GDP and its large
scale movement into the peptidyl transferase center of the
A-site [25,26]. The accommodation step is rate limiting for
peptide-bond formation and at least partially rate limiting
for the entire elongation cycle [27,28]. Because accommodation involves the release of the esterified amino acid
from its specific site on EF-Tu and its entry into the
potentially specific peptidyl transferase center, it seems
likely that the identity of the esterified amino acid could
affect this step.
Whereas a similar rate of accommodation has been
observed for two different cognate aa-tRNAs [27,29],
kinetic measurements of misacylated tRNAs to assess
amino acid specificity at this step are yet to be performed.
However, some data suggest that the ribosomal A-site
itself is specific for binding different amino acid side
chains. Bhuta and coworkers [30] measured the activity of
a series of aminoacylated derivatives of the dinucleotide
CpA in a peptidyl transferase reaction and found that
their Km values depended upon the identity of the amino
acid attached to the CpA derivatives. In addition, Starck
et al. [31] showed that the inhibition efficiency of various
puromycin derivatives depended on the amino acid side
chain of the derivative [31]. Because both of these small
molecule derivatives mimic the 3 0 end of aa-tRNA and
bind at the peptidyl transferase center, these experiments
clearly indicate that the ribosome displays specificity for
different amino acid side chains. Although the data are
limited, the relative affinities of different side chains have
a different hierarchy than found for EF-Tu (Figure 3),
which is consistent with their fitting into a different
binding pocket.
Experiments that measure the binding affinities of
intact aa-tRNAs to the ribosomal A-site are more
ambiguous with respect to amino acid specificity than
experiments using mimics of the 3 0 end of aa-tRNA. The
dissociation rates of eight different tRNAs from the
ribosomal A-site were identical when the tRNAs
were aminoacylated but were quite different when they
were deacylated [32]. This suggests that amino acids make
differing contributions to the affinity of each aa-tRNA for
the A-site and implies that the A-site displays amino acid
specificity. By contrast, several misacylated tRNAs
measured in the same assay displayed dissociation rates
surprisingly similar to their cognate tRNAs [33]. This
either means that the A-site does not show amino acid
specificity or that the specificity is masked by a ribosomal
conformational change that must occur before the aatRNA is released. Clearly, additional binding and kinetic
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663
experiments need to be conducted to better establish the
specificity of the ribosome for different amino acids.
Reconciling previous experiments
Given that EF-Tu and the ribosome seem to show
specificity for the esterified amino acid and the tRNA
body, how can one reconcile the experiments (reviewed
here) that indicate that misacylated tRNAs can function
normally in translation? One suggestion is that, of the
many possible misacylated tRNAs, only certain pairs of
tRNAs and amino acids are likely to show reduced
translational efficiency. Good examples are the successful
incorporation of Ala-tRNACys [3] and poor incorporation of
Glu-tRNAGln [12]. Although their ribosome specificities
are not known, tRNAAla and tRNACys bind to EF-Tu with
similar affinities (Figure 3), indicating that Ala-tRNACys
binds to EF-Tu nearly as well as cognate Ala-tRNAAla, and
could be efficiently delivered to the ribosome. Moreover,
tRNAGlu and tRNAGln, in addition to glutamic acid and
glutamine, have different affinities for EF-Tu (Figure 3),
which leads to the prediction that the misacylated GlutRNAGln binds to EF-Tu weakly. Thus, the binding
properties of EF-Tu for these two misacylated tRNAs
might explain their relative activities in translation. In
other cases, the relative specificity of the ribosome for
amino acids might determine the activity of
misacylated tRNAs.
Even if a misacylated tRNA is reduced in its translational efficiency, it will not necessarily lead to a reduced
overall yield of protein unless it is used at many codons.
For example, at an average amino acid incorporation time
of 50 ms (20 amino acids per second), a protein of 100
amino acids will be elongated in 5 s. If it is assumed that
the same protein must use a misacylated tRNA four times
and that the misacylated tRNA is incorporated 15 times
slower (750 ms), then the same protein will be elongated in
8 s. Such a difference in the incorporation rate of a single
amino acid would be easily detectable by rapid-mixing
experiments [27] and lead to the conclusion that the
misacylated tRNA had a substantially reduced translational efficiency. However, this same difference in rate
might not impact the steady-state levels of the protein
when analyzed in vivo or in an in vitro translation assay.
In other words, global analyses of overall protein
production tend to overestimate the incorporation efficiency of a misacylated tRNA.
A similar argument might explain why so many
misacylated suppressor tRNAs seem to be fully active in
translation. A suppressor tRNA is considered effective if it
can efficiently read a single nonsense codon before the
translation-termination machinery can terminate the
protein. However, because translation termination is
slow compared with a single elongation step [27,34,35], a
suppressor tRNA with a substantially reduced elongation
step can still effectively suppress termination. Thus, a
misacylated tRNA might be active as a suppressor but
might function too poorly to be an active elongator tRNA.
Perspective: an evolving view of tRNA
It has long been known that part of the sequence diversity
among tRNAs results from a need to function in translation
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Vol.30 No.12 December 2005
(a)
Ala-tRNAAla
KD = 6.2nM
Gln-tRNAAla
KD = 0.05nM
Ala-tRNAGln
KD = 260nM
Gln-tRNAGln
KD = 4.4nM
(b)
Amino acids
G lu
tRNAs
WEAK
G lu
Asp Asp
Thr
Ala
Ala
Asp
Leu
G ly
G ly
G ly
Ala
Lys
Val
Cys
Val
M et
Leu
M et
Lys
M et
Arg
Arg
Pro
Thr
Pro
Phe
Pro
Phe
Lys
Phe
Thr
Arg
Ile
Ile
Ser
Ser
Asn
Asn
Val
Tyr
Ile
Cys
Trp
Trp
Trp
G ln
G ln
G ln
STRONG
Tyr
STRONG
WEAK
Ti BS
Figure 3. Thermodynamic compensation by EF-Tu. (a) Experimental affinities of two tRNA bodies esterified with two amino acids [21]. Because the thermodynamic
contributions of the amino acid and the tRNA balance one another, Ala-tRNAAla and Gln-tRNAGln bind to EF-Tu with similar affinities. ‘Weakly’ binding alanine is esterified to a
cognate ‘strongly’ binding tRNAAla, and ‘strongly’ binding glutamine is esterified to a cognate ‘weakly’ binding tRNAGln. Consequently, misacylated Ala-tRNAGln binds to EFTu weakly and Gln-tRNAAla binds to EF-Tu strongly, compared with the cognate aa-tRNAs. (b) Thermodynamic contributions of the different amino acids to EF-Tu$aa-tRNA
binding. Nineteen amino acid side chains (data for histidine was not obtained) display a large range of affinities for EF-Tu. The order of thermodynamic contributions for
amino acid binding to EF-Tu has been determined experimentally for 13 amino acids (bold) [22], and predicted for 19 amino acids (italics) [48]. tRNAs also display a highly
variable range of affinities for EF-Tu [23]. As would be expected from the thermodynamic compensation model, the amino acid and tRNA hierarchies are approximately
inversely proportional, but the correlation is not perfect because cognate aa-tRNAs do not bind to EF-Tu identically [17,18].
in a manner that is specific for each codon–anticodon pair.
Residues in the anticodon stem-loop correlate with the
identity of the anticodon [36], and mutations of those
residues affect the translatability of tRNA [37,38]. In
addition, many of the diverse post-transcriptional modifications in tRNA subtly affect translational efficiency when
they are deleted [39–41]. Recently, we suggested that this
idiosyncratic evolutionary ‘tuning’ of tRNAs is required to
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permit uniform tRNA function in translation [42]. In other
words, part of the sequence and modification diversity of
tRNAs is to compensate for the structural, thermodynamic
and kinetic differences that arise from the need for different
codon–anticodon pairs to be accommodated in translation.
Here, we have discussed data which suggest that an
additional evolutionary source of tRNA sequence and
modification diversity is the identity of the esterified
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TRENDS in Biochemical Sciences
amino acid. As a result of the environment of the aminoacid-binding pockets of EF-Tu and the ribosome, each
amino acid has distinct binding properties. As a consequence of the need for uniformity, the sequences of the
tRNA bodies have evolved to compensate for the amino
acid specificity of the translational machinery. Thus,
tRNAs use diversity to function uniformly.
In summary, mounting evidence [1,29,36] demands
that the traditional view of tRNAs as interchangeable,
‘passive’ adaptors be discarded. Instead, one must think of
tRNAs as active participants in translation that have
individually evolved to meet the idiosyncratic needs
dictated by their amino acid and anticodon.
Acknowledgements
This work was supported by National Institutes of Health Grant
GM37552 to O.C.U.
References
1 Woese, C.R. (2001) Translation: in retrospect and prospect. RNA 7,
1055–1067
2 Crick, F.H. (1958) On protein synthesis. Symp. Soc. Exp. Biol. 12,
138–163
3 Chapeville, F. et al. (1962) On the role of soluble ribonucleic acid in
coding for amino acids. Proc. Natl. Acad. Sci. U. S. A. 48, 1086–1092
4 Prather, N.E. et al. (1984) Nucleotide substitution in the amino acid
acceptor stem of lysine transfer RNA causes missense suppression.
J. Mol. Biol. 172, 177–184
5 Normanly, J. et al. (1990) Construction of Escherichia coli amber
suppressor tRNA genes. III. Determination of tRNA specificity. J. Mol.
Biol. 213, 719–726
6 Tsai, F. and Curran, J.F. (1998) tRNA(2Gln) mutants that translate
the CGA arginine codon as glutamine in Escherichia coli. RNA 4,
1514–1522
7 Saks, M.E. et al. (1994) The transfer RNA identity problem: a search
for rules. Science 263, 191–197
8 Giegé, R. et al. (1998) Universal rules and idiosyncratic features in
tRNA identity. Nucleic Acids Res. 26, 5017–5035
9 Wang, L. and Schultz, P.G. (2004) Expanding the genetic code. Angew.
Chem. Int. Ed. Engl. 44, 34–66
10 Ibba, M. et al. (2000) The adaptor hypothesis revisited. Trends
Biochem. Sci. 25, 311–316
11 Ibba, M. and Söll, D. (2000) Aminoacyl-tRNA synthesis. Annu. Rev.
Biochem. 69, 617–650
12 Stanzel, M. et al. (1994) Discrimination against misacylated tRNA by
chloroplast elongation factor Tu. Eur. J. Biochem. 219, 435–439
13 Kobayashi, T. et al. (2005) Structural snapshots of the KMSKS loop
rearrangement for amino acid activation by bacterial tyrosyl-tRNA
synthetase. J. Mol. Biol. 346, 105–117
14 Nissen, P. et al. (1995) Crystal structure of the ternary complex of
Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 270, 1464–1472
15 Schmeing, T.M. et al. (2002) A pre-translocational intermediate in
protein synthesis observed in crystals of enzymatically active 50S
subunits. Nat. Struct. Biol. 9, 225–230
16 Nissen, P. et al. (1999) The crystal structure of Cys-tRNACys-EF-TuGDPNP reveals general and specific features in the ternary complex
and in tRNA. Structure Fold. Des. 7, 143–156
17 Louie, A. and Jurnak, F. (1985) Kinetic studies of Escherichia coli
elongation factor Tu-guanosine 5 0 -triphosphate-aminoacyl-tRNA
complexes. Biochemistry 24, 6433–6439
18 Ott, G. et al. (1990) Ternary complexes of Escherichia coli aminoacyltRNAs with the elongation factor Tu and GTP: thermodynamic and
structural studies. Biochim. Biophys. Acta 1050, 222–225
19 Pingoud, A. and Urbanke, C. (1980) Aminoacyl transfer ribonucleic
acid binding site of the bacterial elongation factor Tu. Biochemistry 19,
2108–2112
20 Wagner, T. and Sprinzl, M. (1980) The complex formation between
Escherichia coli aminoacyl-tRNA, elongation factor Tu and GTP. The
effect of the side-chain of the amino acid linked to tRNA. Eur.
J. Biochem. 108, 213–221
www.sciencedirect.com
Vol.30 No.12 December 2005
665
21 LaRiviere, F.J. et al. (2001) Uniform binding of aminoacyl-tRNAs to
elongation factor Tu by thermodynamic compensation. Science 294,
165–168
22 Dale, T. et al. (2004) The affinity of elongation factor Tu for an
aminoacyl-tRNA is modulated by the esterified amino acid. Biochemistry 43, 6159–6166
23 Asahara, H. and Uhlenbeck, O.C. (2002) The tRNA Specificity of
Thermus thermophilus EF-Tu. Proc. Natl. Acad. Sci. U. S. A. 99,
3499–3504
24 Blanchard, S.C. et al. (2004) tRNA selection and kinetic proofreading
in translation. Nat. Struct. Mol. Biol. 11, 1008–1014
25 Stark, H. et al. (2002) Ribosome interactions of aminoacyl-tRNA and
elongation factor Tu in the codon-recognition complex. Nat. Struct.
Biol. 9, 849–854
26 Valle, M. et al. (2003) Incorporation of aminoacyl-tRNA into the
ribosome as seen by cryo-electron microscopy. Nat. Struct. Biol. 10,
899–906
27 Pape, T. et al. (1998) Complete kinetic mechanism of elongation factor
Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli
ribosome. EMBO J. 17, 7490–7497
28 Savelsbergh, A. et al. (2003) An elongation factor G-induced ribosome
rearrangement precedes tRNA-mRNA translocation. Mol. Cell 11,
1517–1523
29 Cochella, L. and Green, R. (2005) An active role for tRNA in decoding
beyond codon:anticodon pairing. Science 308, 1178–1180
30 Bhuta, A. et al. (1981) Stereochemical control of ribosomal peptidyltransferase reaction. Role of amino acid side-chain orientation of
acceptor substrate. Biochemistry 20, 8–15
31 Starck, S.R. et al. (2003) The puromycin route to assess stereo- and
regiochemical constraints on peptide bond formation in eukaryotic
ribosomes. J. Am. Chem. Soc. 125, 8090–8091
32 Fahlman, R.P. et al. (2004) Uniform binding of aminoacylated transfer
RNAs to the ribosomal A and P sites. Mol. Cell 16, 799–805
33 Dale, T. and Uhlenbeck, O.C. Binding of misacylated tRNAs to the
ribosomal A site. RNA (in press)
34 Zavialov, A.V. et al. (2002) Release of peptide promoted by the GGQ
motif of class 1 release factors regulates the GTPase activity of RF3.
Mol. Cell 10, 789–798
35 Peske, F. et al. (2005) Sequence of steps in ribosome recycling as
defined by kinetic analysis. Mol. Cell 18, 403–412
36 Yarus, M. (1982) Translational efficiency of transfer RNA’s: uses of an
extended anticodon. Science 218, 646–652
37 Yarus, M. et al. (1986) The translational efficiency of tRNA is a
property of the anticodon arm. J. Biol. Chem. 261, 10496–10505
38 Raftery, L.A. and Yarus, M. (1987) Systematic alterations in the
anticodon arm make tRNA(Glu)-Suoc a more efficient suppressor.
EMBO J. 6, 1499–1506
39 Urbonavicius, J. et al. (2001) Improvement of reading frame
maintenance is a common function for several tRNA modifications.
EMBO J. 20, 4863–4873
40 Urbonavicius, J. et al. (2002) Three modifications in the D and T arms
of tRNA influence translation in Escherichia coli and expression of
virulence genes in Shigella flexneri. J. Bacteriol. 184, 5348–5357
41 Nasvall, S.J. et al. (2004) The modified wobble nucleoside uridine-5oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four
proline codons in vivo. RNA 10, 1662–1673
42 Olejniczak, M. et al. (2005) Idiosyncratic tuning of tRNAs to achieve
uniform ribosome binding. Nat. Struct. Mol. Biol. 12, 788–793
43 Ogle, J.M. et al. (2003) Insights into the decoding mechanism from
recent ribosome structures. Trends Biochem. Sci. 28, 259–266
44 Ogle, J.M. and Ramakrishnan, V. (2005) Structural insights into
translational fidelity. Annu. Rev. Biochem. 74, 129–177
45 Rould, M.A. et al. (1989) Structure of E. coli glutaminyl-tRNA
synthetase complexed with tRNA(Gln) and ATP at 2.8 Å resolution.
Science 246, 1135–1142
46 Polekhina, G. et al. (1996) Helix unwinding in the effector region of
elongation factor EF-Tu–GDP. Structure 4, 1141–1151
47 Hansen, J.L. et al. (2003) Structures of five antibiotics bound at the
peptidyl transferase center of the large ribosomal subunit. J. Mol.
Biol. 330, 1061–1075
48 Asahara, H. and Uhlenbeck, O.C. (2005) Predicting the binding
affinities of misacylated tRNAs for Thermus thermophilus EFTu.GTP. Biochemistry 44, 11254–11261