From the Département "Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse," UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, F-67084 Strasbourg Cedex, France
Received for publication, October 29, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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This study evaluates the role of the N-terminal
extension from yeast aspartyl-tRNA synthetase in tRNA aspartylation.
The presence of an RNA-binding motif in this extension, conserved in
eukaryotic class IIb aminoacyl-tRNA synthetases, provides nonspecific
tRNA binding properties to this enzyme. Here, it is assumed that the additional contacts the 70 amino acid-long appendix of aspartyl-tRNA synthetase makes with tRNA could be important in expression of aspartate identity in yeast. Using in vitro transcripts
mutated at identity positions, it is demonstrated that the extension
grants better aminoacylation efficiency but reduced specificity to the synthetase, increasing considerably the risk of noncognate tRNA mischarging. Yeast tRNAGlu(UUC) and
tRNAAsn(GUU) were identified as the most easily mischarged
tRNA species. Both have a G at the discriminator position, and their
anticodon differs only by one change from the GUC aspartate anticodon.
Aminoacylation of tRNA is a key step during translation of genetic
information. It is performed by aminoacyl-tRNA synthetases (aaRSs)1 and involves
specific recognition of their cognate tRNAs and amino acids (reviewed
in Ref. 1). Fidelity of translation depends on accurate tRNA
aminoacylation, but unexpectedly, this reaction does not occur with
absolute specificity (2). This dilemma is resolved since synthetases
developed kinetic artifices ensuring destruction of the mischarged
tRNAs (editing), thereby reducing error levels in protein synthesis
(3). Furthermore, specialized proteins, by interacting with certain
synthetases, increase their affinity for the cognate tRNA and thus
ensure increased aminoacylation efficiency. This is the case in the
association of Arc1p with MetRS and GluRS in yeast (4) and of p43 with
ArgRS in mammals (5).
A similar "helper" role seems to be played by the extra domains
within eukaryotic synthetases. Indeed, these enzymes differ usually
from their prokaryotic counterparts by appendices located in their N-
or C-terminal regions (6), outside the structural core defining the two
classes of synthetases (7). In mammals, the extensions are involved in
the formation of the multi-synthetase complex (6), but such
architecture has not been detected in unicellular eukaryotes until now.
In fact, little is known about the functional role of the extensions in
unicellular eukaryotic synthetases, except that in two different yeast
aminoacylation systems, they were shown to provide nonspecific tRNA
binding properties to GlnRS (8, 9) and AspRS (10).
Yeast AspRS contains an N-terminal appendix of 70 amino acids
that adopts a helical structure (11, 12). It encompasses an RNA-binding
motif (XSKXXLKKXK) conserved in
eukaryotic AspRSs but also in eukaryotic LysRSs and AsnRSs (10), the
two other class IIb synthetase families. The appendix, located next to
the anticodon-binding module, was shown to considerably increase the stability of the complex between AspRS and cognate tRNAAsp
as shown by gel-shift binding assays and footprinting experiments. Moreover, the AspRS appendix can be replaced by the appendices of LysRS
or AsnRS (10). Likewise, it was recently demonstrated that the
extension in human LysRS shares the same properties of those in yeast
AspRS (13). However, its presence in AspRS is not mandatory for tRNA
aminoacylation (11, 14). As seen in the crystal structure of yeast
AspRS complexed with tRNAAsp (15), and in that recently
solved of the free enzyme truncated of its first 70 amino acids (16),
only the core of the enzyme is required for tRNA aspartylation.
Specificity of tRNA recognition by yeast AspRS is primarily ensured by
the tRNA identity determinants. Only six such determinants, located in
three distinct regions of tRNAAsp (Fig.
1A), are important to confer
the aspartate identity. They are the discriminator base G73, four bases
in the anticodon loop (G34, U35, C36, and C38), and base pair G10-U25
in the core region of the tRNA (17, 18). Mutation of these elements
leads to losses in aspartylation efficiencies (17), the strongest
determinants being those leading to the strongest effects upon mutation
(e.g. G73 and the aspartate anticodon G34, U35, C36). Except
for identity base pair G10-U25, which is involved in the correct
folding of the tRNA (19) and has no direct contact with the enzyme, all other identity bases make specific interactions with amino acid residues on AspRS (20). Nonspecific contacts, however, are much more
numerous. As shown by biochemical and x-ray data, they extend along the
side of the tRNA comprising the variable region (15, 21, 22) (Fig.
1B, left). Additional nonspecific contacts are provided by the N-terminal extension of native AspRS and are found along the opposite side of the tRNA (Fig. 1B,
right). They increase the stability of the complex and the
global aminoacylation efficiency (10).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Structure of yeast tRNAAsp
with AspRS recognition elements emphasized. A, cloverleaf
structure of tRNAAsp. Identity determinants recognized
specifically by AspRS are circled in red.
B, structure of the AspRS/tRNAAsp complex as
determined in the crystal structure (left) and modelization
of the N-terminal appendix (right) (adapted from Refs. 15
and 10, respectively). The N-terminal extension was modeled in
blue, and the RNA-binding motif is indicated in
pink.
Considering the better affinity of tRNAAsp for native AspRS
and the increased aspartylation efficiency correlated with the presence of the N-terminal extension in the native enzyme, it can be conjectured that the additional contacts between the two macromolecular partners play a role in the mechanisms ensuring the specificity of genetic code
expression at the translational level. By mutational analysis of
tRNAAsp and AspRS, it is shown that the N-terminal
extension is responsible for a decrease in aminoacylation specificity,
thereby favoring aspartylation of noncognate tRNAs. The identified
mischarged tRNAs are related with tRNAAsp by their
anticodon sequence, in agreement with the concept of partial
conservation of determinants in mischarged tRNAs. Altogether, a
mechanistic role of the yeast AspRS N-terminal extension in the
aspartylation reaction of tRNA is established. This has functional implications for the specific expression of aspartate identity in
vivo.
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EXPERIMENTAL PROCEDURES |
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Preparation of Pure AspRSs and of Crude Synthetase Extracts
Native AspRS1-557 from yeast and truncated
AspRS71-557, deleted of its N-terminal extension, were
expressed and purified as described previously (10). In short, the
genes were cloned into the Escherichia coli expression
vector pQE70 (Qiagen) fused to a C-terminal His6 tag. AspRS
variants were purified according to the Qiagen protocol, quantified by
Bradford assay (Bio-Rad), and stored at 20 °C in 50 mM
phosphate buffer, pH 7.2, 150 mM KCl, 50% glycerol, and 10 mM
-mercaptoethanol. AspRS preparations yielded only one
band on SDS-polyacrylamide gel, even when overloaded, and can be
estimated to be 99% pure. Specific activities, expressed as turnover
numbers, correspond to 0.20 and 0.19 s
1 for
AspRS1-557 and AspRS71-557, respectively
(assays conducted in the presence of 25 µM aspartate, a
subsaturating amino acid concentration, see Ref. 23) or to 3.4 and 3.1 s
1 for AspRS1-557 and
AspRS71-557, respectively (assays conducted in the
presence of 500 µM aspartate, a concentration close to
the physiological level of aspartate in yeast cells, see Ref.
24). An enriched protein extract deprived of nucleic acids and
ribosomes was made by chromatography on a Mono-Q column (Bio-Rad) and
used as a crude enzyme preparation containing the activity of
aminoacyl-tRNA synthetases (except for prolylation and glutaminylation).
Preparation of tRNAs
In Vitro Transcription of tRNAAsp Variants-- tRNA transcripts used in this work have been obtained by in vitro transcription of synthetic genes cloned in pUC 118 (17). Each of these genes corresponds to the T7 RNA polymerase promoter region directly connected to the downstream tRNA sequence. In vitro transcriptions were performed in reaction mixtures containing 40 mM Tris-HCl, pH 8.1 (37 °C), 22 mM MgCl2, 5 mM dithioerythritol, 0.1 mM spermidine, 4 mM each of nucleoside triphosphate, 5 mM GMP, 50 ng/µl linearized plasmid, and 5 µg/ml T7 RNA polymerase. Transcription mixtures were incubated for 2 h at 37 °C, and reactions were stopped by phenol/chloroform extraction. Full-length transcripts correctly ending with the CCA sequence were purified by preparative electrophoresis on 12% polyacrylamide denaturing gels (8 M urea) followed by electroelution in Tris-borate-EDTA (Schleicher and Schuell apparatus). The concentration of tRNA transcripts was determined by absorbance at 260 nm.
Purification of Yeast tRNAs-- Countercurrent fractions enriched in tRNAAsn, tRNAAsp or tRNAGlu were gifts from G. Keith. Pure tRNAAsn and tRNAGlu were obtained by purification on a 10% polyacrylamide gel (19/1), in semidenaturing conditions (4 M urea). Migration was performed at 300 V for 48 h at 4 °C. Bands containing pure tRNAs were handled as described for tRNA transcripts. Pure tRNAAsp was a gift from A. Théobald-Dietrich. The purity of tRNAs (>95%) was established by testing their cognate amino acid acceptance.
Aminoacylation of tRNAs
Aminoacylations of tRNAAsp variants have been performed at 37 °C in 100 mM HEPES-KOH, pH 7.4, 15 mM MgCl2, 5 mM ATP, 30 mM KCl, 25 µM L-[3H]aspartate (Amersham Biosciences), and appropriate amounts of tRNA transcripts. Before aminoacylation, transcripts were renatured in H2O by heating at 85 °C for 90 s and slow cooling to room temperature prior adjunction of MgCl2. Aminoacylation reactions were initiated by addition of appropriate amounts of enzyme diluted in 100 mM HEPES-KOH, pH 7.4, 10% glycerol, 1 mM dithioerythritol, and 5 mg/ml bovine serum albumin. Aminoacylated tRNA samples were quenched in trichloroacetic acid and treated by the conventional way on Whatman paper 3 MM (25). Kinetic constants (kcat and Km) were derived from Lineweaver-Burk plots. In general, the concentration of tRNA transcripts ranged from 0.1 to 5 Km when possible, except for mutants with Km over 10 µM, where the highest concentration was at most 2-3 Km. Plateaus of tRNA aspartylation were determined by incubating 1 µM pure tRNA (transcript or modified species) or 20 µM total tRNA in the presence of different AspRS (0.05-5 µM) and aspartate (25-500 µM) concentrations and otherwise optimal reaction conditions (as above). Reactions were stopped after 2.5, 5, 10, and 20 min incubation at 37 °C. Displayed kinetics and plateaus represent an average of at least two independent experiments. Values of kcat/Km ratios for replicate experiments varied by at most 15%.
Identification of Mischarged tRNAs
Total Yeast tRNA-- The brewers' yeast tRNA was from Roche Molecular Biochemicals. Titration by aminoacylation assays, using crude synthetases extract and 25 µM aspartate, indicated a content of 4.5 ± 0.5% tRNAAsp in this total tRNA (deduced from plateau values).
Protection of Thiolated Bases-- To avoid modification of thiolated bases during periodate oxidation (26), the total yeast tRNA was DTNB-treated as follows. Total tRNA (20,000 pmol) was incubated for 20 min at room temperature in 25 mM phosphate buffer, pH 6.8, in the presence of 0.3 mM DTNB (Sigma). DTNB was removed by dialyzing 12 h against milliQ water, and tRNA was recovered by precipitation.
Aspartylation Reactions-- Total tRNA (20 µM, containing ~1 µM tRNAAsp) was incubated with 10 µM native or truncated AspRS in the presence of 500 µM aspartic acid (ratio [AspRS]/[tRNAAsp] = 5). Aminoacylation was conducted at 37 °C for 25 min; it was stopped by phenol extraction (pH 5.0), and the tRNA was recovered by ethanol precipitation. Controls without AspRS were run in parallel.
Oxidation of Non-aspartylated tRNAs-- The periodate treatment of tRNA was adapted from Hansske and Cramer (27). The pellet containing charged and uncharged tRNAs was dissolved in 50 mM sodium acetate buffer, pH 5.0, to a final concentration of 80 µM. Oxidation was started by addition of 40 mM m-periodate (Sigma) and was run at room temperature in the dark for 25 min. The reaction was stopped by neutralizing the excess periodate with 5% glycerol. Glycerol and remaining periodate were removed by dialysis (2 h against 5 mM sodium acetate at pH 5.0). The mixture of aspartylated and of oxidized tRNA species was recovered by ethanol precipitation.
Deacylation and Deprotection of tRNAs-- The tRNA pellet was dissolved in 1.8 M Tris-HCl, pH 8.0, to a final concentration of 80 µM and incubated for 30 min at 37 °C for complete deacylation of aspartylated tRNA species. After precipitation, tRNAs were first incubated for 3 h on ice in 0.1 M dithioerythritol (reduction of thiol groups) and for 20 min at room temperature in 0.1 M NaBH4 (reduction of periodate-generated aldehyde groups on the terminal ribose of non-aspartylated tRNAs). NaBH4 was removed by an overnight dialysis in milliQ water. tRNAs were recovered by ethanol precipitation and quantified by absorbance at 260 nm.
Identification of Aspartylated/Protected
tRNAs--
Three batches of partially aminoacylated tRNAs (a control
and two batches that were aspartylated by native
AspRS1-557 or truncated AspRS71-557) were
tested for their amino acid acceptance. Aminoacylation activities were
measured under the same conditions as those described above, with 25 µM amino acid, 20 µM treated tRNA, and 0.15 µg/µl crude enzymatic extract containing aminoacyl-tRNA synthetase
activities. Eighteen amino acids were tested:
L-[14C]-radiolabeled amino acids were
alanine, arginine, glutamic acid, glutamine, histidine, isoleucine,
leucine, methionine, phenylalanine, proline, serine, tyrosine, and
valine; those [3H]-labeled were asparagine, aspartic
acid, glycine, lysine, and threonine (all from Amersham Biosciences).
Purity of [3H]-labeled aspartic acid and asparagine as
well as of cold aspartic acid, asparagine, and glutamic acid was
checked by TLC for possible contamination with other amino acids. No
visible trace of such contamination has been detected.
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RESULTS AND DISCUSSION |
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The N-terminal Extension Confers Increased Aminoacylation Efficiency but Decreased Specificity to AspRS-- Aspartylation efficiencies of tRNAAsp transcripts mutated at their major identity positions (the discriminator base G73 and the anticodon bases G34, U35, C36) were monitored. For each mutant, the kinetic constants (kcat and Km) were derived from aminoacylation reactions with the truncated and native AspRS (AspRS71-557 and AspRS1-557) (Table I). For each AspRS form, aminoacylation efficiencies (kcat/Km) were calculated for mutated tRNAs. For an easier comparison, results will be discussed as losses of aminoacylation efficiency calculated as (kcat/Km)wild-type/(kcat/Km)mutant. The higher the losses of aminoacylation efficiency, the better the discrimination control of the synthetase toward the mutated position in tRNAAsp.
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As shown previously, kinetic constants determined for aspartylation of fully modified tRNAAsp are only slightly different from those measured for the corresponding wild-type transcript (25). For truncated AspRS, both kcat and Km of the aspartylation reaction stay almost the same. With native AspRS, only the Km of the modified tRNAAsp is lowered ~17 times as compared with the wild-type transcript (Table I).
With truncated AspRS71-557, mutations at single identity
positions in tRNA transcripts have strong effects on the global
aminoacylation efficiency, which can be reduced up to ~3000-fold. At
position 35, replacement of the wild-type U by any other base leads to sharp decreases in aminoacylation efficiency (between 700- and 1400-fold). At the three other major identity positions, 73, 34, and
36, all mutations except one lead to lower decreases of aminoacylation efficiencies with effects fluctuating between 4- and 340-fold depending
on the mutation introduced. Noteworthily, the G34 C exception is
the mutation leading to the strongest effect (~3000-fold). In
general, comparison of the individual kinetic constants indicates that
Km is the most affected (exceptions are mutations G73
A and G34
C where both kcat and
Km are changed).
The same experiments, done with native AspRS1-557 under
identical conditions, gave significantly different results. Although as expected, mutations of tRNAAsp identity positions still
cause decreases in aspartylation efficiency, their effects are
quantitatively and qualitatively changed. Indeed, with the native
synthetase, the losses of aminoacylation efficiency reach at most
~500-fold (as compared with the ~3000-fold decrease for the
strongest loss with AspRS71-557), and the involved
positions are different (e.g. strongest effect for U35 G
with native AspRS1-557 and for G34
C with truncated
AspRS71-557). For 8 out of 12 mutants, losses in
aminoacylation efficiency varies between 1- and 22-fold, and only four
tRNAAsp mutants are poorly aminoacylated, with losses in
aminoacylation efficiencies above 80. They correspond to transcripts
containing mutations G73
C, G34
C, or U35
A or G. Here
again, affinity (reflected by Km) between native
AspRS1-557 and the tRNA substrates is mostly affected,
whereas kcat is only moderately affected.
These kinetic differences observed in aspartylation reactions conducted
with native or truncated AspRSs have dramatic consequences. To evaluate
these consequences, the gain in aminoacylation efficiency consecutive
to the presence of the N-terminal extension in AspRS was calculated for
each tRNA variant, comparing their aminoacylation efficiencies between
both native and truncated AspRSs. Whatever the tRNA substrates,
wild-type or mutants, the aminoacylation efficiencies are strongly
stimulated when the N-terminal extension is present in AspRS. The
strengths of these positive effects, however, are dependent on
the mutation introduced in the tRNA. As seen in Table I, these values
comprise between 3 and 171. Indeed, the N-terminal appendix triggers
only a moderate increase of the aminoacylation efficiency of
tRNAAsp transcripts mutated at the discriminator position
(at most 11-fold for the G73 U mutation). In contrast, its effect
is increased at least by one order of magnitude when the mutations are
introduced in the anticodon (up to ~170-fold for the U35
C mutation).
Altogether, this means that native AspRS1-557 is less discriminative than truncated AspRS71-557. This property of native AspRS1-557, a priori unexpected, is solely due to the presence of the N-terminal extension. This finding shows that the role of the extension is not marginal and has functional consequences.
Effects of Degradations in AspRS Extension on tRNA Charging-- Globally, the data in Table I confirm the mutational analysis on tRNA aspartate identity determinants obtained previously with another AspRS sample (17), but they diverge quantitatively. The differences rely on the chemical nature of the enzymes used in the two studies. The synthetase used formerly was purified directly from Saccharomyces cerevisiae, whereas the two enzymes used in the present study were cloned and overexpressed in E. coli. It was known that during purification from yeast, AspRS is subjected to partial proteolysis within the RNA-binding motif in its N-terminal extension (10, 11) with the consequence that the purified synthetase is a heterogeneous protein pool. Thus, the overall effects found with the degraded enzyme (17) are intermediary with those obtained with native AspRS1-557 and truncated AspRS71-557 with aminoacylation efficiencies predominantly kcat-directed, in contrast to the present report where they are mainly Km-directed.
Functional Implications--
As a consequence of the presence of
the N-terminal extension in AspRS, the anticodon identity nucleotides
are recognized with rather low discrimination (Table I). Since
identity of a tRNA primarily relies on the presence of the major
determinants rather than on the sequence context in which they are
embedded (28), this implies that in a native tRNA pool, molecules
having anticodon sequences close to the GUC aspartate anticodon should
be recognized by native AspRS1-557 with efficiencies close
to that of cognate tRNAAsp. Several yeast tRNA isoacceptors
answer this prerequisite, namely tRNAAsn(GUU),
tRNAGlu(UUC), tRNAHis(GUG),
tRNATyr(GUA), tRNAGly(GCC),
tRNAAla(GGC), and tRNAVal(GAC). Among them,
tRNAHis, tRNATyr, tRNAGly,
tRNAAla, and tRNAVal could be reasonably
eliminated because their discriminator base is A, instead of G as in
tRNAAsp. Indeed, it was shown that two mutations in
tRNAAsp transcripts distributed between the anticodon and
the discriminator position lead to cooperative effects that decrease
severely aspartylation (29). Considering these assumptions, only
tRNAAsn(GUU) and tRNAGlu(UUC) are left as
good candidates for misacylation by AspRS (Fig.
2).
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The Yeast AspRS Extension Increases the Risk of tRNA
Mischarging--
To determine the total amount of tRNA species that
can be aspartylated, total yeast tRNA was incubated in the presence of different concentrations of native and truncated AspRSs and under saturating aspartate concentrations (500 µM). Five
aminoacylation conditions were tested with a fixed concentration of
total tRNA (20 µM) and increasing concentrations of
synthetases (from 0.125 to 5 µM). Thus, molar ratios
between the tRNAAsp fraction present in total yeast tRNA
(4.5%, see "Experimental Procedures") and AspRS were 1/0.125,
1/0.5, 1/1.25, 1/2.5, and 1/5. When ratios of
tRNAAsp/AspRS1-557 or
tRNAAsp/AspRS71-557 were 1/0.5, both enzymes
show the same charging capacity with 6% of total tRNA aspartylated.
This level reflects aspartylation of part of tRNAAsp
present in total yeast tRNA (~4.5%) plus a significant amount of
mischarged tRNA species (~1.5%). The amount of aspartylated tRNA
increases with the AspRS concentration in the aminoacylation assays.
However, the raise is much more pronounced in assays conducted with
native AspRS1-557 (Fig. 3).
Thus, when present at a 5-fold molar excess as compared with
tRNAAsp, the amount of aspartylated tRNA catalyzed by
native AspRS1-557 is more than 2-fold of that of
tRNAAsp, whereas with truncated AspRS71-557,
it reaches only 1.5-fold. This indicates that AspRS aspartylates noncognate tRNA(s) and that the level of mischarging is significantly higher with native AspRS1-557. In control experiments
conducted in the presence of limiting aspartate concentration (25 µM), the mischarging potential of AspRS, even in its
native version, is suppressed (Fig. 3).
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Identification of Aspartylated Noncognate tRNAs-- At first, to prevent false interpretation of results, care was taken that AspRS preparations as well as amino acids were of enough purity to avoid possible misaminoacylation catalyzed by trace contaminations of E. coli synthetases (see "Experimental Procedures"). The mischarged tRNA species were identified in a three-step procedure. First, total tRNA was aspartylated by either native AspRS1-557 or truncated AspRS71-557 in assays conducted with 500 µM aspartate, i.e. under conditions where the specific activity of both AspRSs is the highest. Further, the AspRS concentration was in 5-fold molar excess as compared with the amount of tRNAAsp present in total yeast tRNA. Second, the pool of aspartylated tRNA (comprising tRNAAsp and putative mischarged noncognate tRNAs) was periodate-treated to inactivate all uncharged tRNAs by oxidation of their 3'-terminal ribose. This treatment does not affect the aspartylated species protected by the aspartate residue esterified on their 3'-end. Before treatment, care was taken to protect tRNAs containing thiolated bases against inactivation by periodate (see "Experimental Procedures"), i.e. yeast tRNAGlu, a potential candidate for mischarging, which contains a 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) residue in its anticodon (30). Third, the periodate-treated tRNA, still containing the pool of aspartylated tRNA species, was deacylated, and formerly mischarged species were identified by their original amino acid acceptance (see "Experimental Procedures").
Aminoacylation of total yeast tRNA with both truncated AspRS71-557 or native AspRS1-557 leads to quantitatively different, although qualitatively comparable, results. As seen in Table II, besides tRNAAsp, three families of noncognate aspartylatable tRNA species can be identified. The first one comprises, as anticipated, tRNAAsn and tRNAGlu and corresponds to molecules abundantly aminoacylated. The second family, with tRNAHis, tRNALys, and tRNAArg, comprises molecules moderately aminoacylated. The third family encompasses molecules that were not mischarged, or if so, mischarged to trace levels.
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With truncated AspRS71-557, the amount of aspartylated tRNA was only slightly above the predicted level of tRNAAsp charging (6.25% versus 4.5%). As expected, tRNAAsp was the major species identified, representing 40% of the charged tRNAs. The 60% tRNAs left were mischarged species. Surprisingly, tRNAGlu and tRNAAsn were mischarged to comparable levels representing each as much as half of the charged tRNAAsp. The minor species correspond to tRNAs mischarged to plateau levels comprising between 1 and 8%.
With native AspRS1-557, as a consequence of the presence of the N-terminal extension, ~50% more tRNA was aspartylated (Table II). The same tRNA species were identified in the aspartylated pool, but they were charged to a higher level. Altogether, aspartylation by the synthetase comprising the extension reaches 74% of the amount of tRNAAsp present in total yeast tRNA, 70% of tRNAGlu, 35% of tRNAAsn, and 3-9% of miscellaneous tRNAs. At this point, it is important to remember that E. coli GluRS cannot charge yeast tRNAGlu (31) and that prokaryotic AsnRSs do not recognize eukaryotic tRNAAsn species (reviewed in Ref. 32). Considering these facts, we can conclude that charging of both tRNAGlu and tRNAAsn did not occur as a consequence of contaminating amino acids and E. coli GluRS and AsnRS present in yeast AspRS preparations.
The two major misacylated tRNAs, namely tRNAGlu and tRNAAsn, both have a G at the discriminator position (as in tRNAAsp), and their anticodon differs only by one base change from the GUC aspartate anticodon (U34 in tRNAGlu(UUC) and U36 in tRNAAsn(GUU)). The less efficient charging of tRNAHis and tRNALys can be explained by less conservation of aspartate identity elements in these tRNAs (only U35 and G73 in tRNALys(UUU) and G34 and U35 in tRNAHis(GUG)). The moderate mischarging of arginine-specific tRNA is more difficult to understand but could be accounted by the sole presence of G73.
Aspartylation of Pure tRNAAsn and tRNAGlu-- Pure fully modified tRNAAsn(GUU) and tRNAGlu(UUC) from S. cerevisiae were tested in mischarging assays with native AspRS1-557 and their aspartylation capacity as compared with that of tRNAAsp. Mischarging plateaus were determined in the presence of low (0.05 µM) and high (5 µM) AspRS1-557 concentrations (data not shown). The results confirm the experiments conducted with total yeast tRNA. At a low AspRS1-557 concentration, only tRNAAsp is charged to a high level (~80%); mischarging of tRNAGlu did not exceed a plateau of 20%, and no aspartylation of tRNAAsn was detected. On the contrary, as soon as the AspRS concentration was increased, 100% of tRNAAsp was charged, and excessive mischarging was detected with plateaus reaching 75% of tRNAGlu and 30% of tRNAAsn aspartylated.
Besides conservation of at least three of the four major aspartate identity elements in tRNAGlu (G73, U35, and C36) and in tRNAAsn (G73, G34, and U35), one finds also in these tRNAs large sequence homologies with tRNAAsp (Fig. 2). In tRNAGlu, they comprise, among others, residue C38, which was shown to influence aspartylation efficiency (18). However, differences appear at the level of the post-transcriptional modifications (Fig. 2). In tRNAGlu, only residue U34 in the anticodon loop is hypermodified (mcm5s2U34), whereas in tRNAAsn, four specific modifications are spread all over its L-shaped structure (m2G10 and m22G26 in the core, t6A37 in the anticodon loop, and m1A58 in the T-loop). In both tRNAs, these modifications could influence negatively their recognition by AspRS. Nevertheless, both tRNAGlu and tRNAAsn become significantly aspartylated when the AspRS concentration is increased, which seems to be sufficient to overcome the slight sequence differences that exist between them and tRNAAsp. Thus, a high AspRS concentration is a threat for accurate expression of yeast aspartate identity.
General Conclusion-- During protein synthesis, errors in amino acid incorporation have been detected (33) that are tolerated by the cell if their level is low enough. Mischarging of tRNA was considered as an obvious cause leading to such errors. It can originate as the result either of misactivated amino acids transferred to the cognate tRNA or of noncognate tRNA misacylations. Different mechanisms have been considered to overcome these errors. Editing is an option for synthetases having low specificity toward their cognate amino acid, as E. coli IleRS (34) and ValRS (35) and its structural foundation explicitly established for prokaryotic IleRS (36) and ThrRS (37). Correction or prevention of tRNA mischarging due to misrecognition of tRNA is less documented. The presence of antideterminants in tRNAs, responsible for blocking noncognate aminoacylation, has been studied. For example, methylation of G37 in yeast tRNAAsp blocks arginylation (38).
Here, in both tRNAGlu and tRNAAsn, base modifications could influence negatively their recognition by AspRS, but they are not sufficient to repress mischarging with aspartate in vitro. In fact, the biological consequences of this kind of mischarging was considered as marginal because it is further repressed in the presence of the cognate tRNA (reviewed in Ref. 39), and this view is strengthened by the observation that in vivo competitions between different synthetases lower the risk of mischarging (40). However, this study demonstrates that yeast AspRS is able to aspartylate in vitro noncognate tRNAs to unexpectedly high levels, even in total tRNA samples containing cognate tRNAAsp. This property is considerably enhanced when the N-terminal extension of the enzyme is present. High mischarging relies on the inherent low specificity of yeast AspRS for its tRNAAsp substrate, as shown by the weak catalytic discrimination toward tRNAAsp molecules mutated at identity positions. This low discrimination is mainly due to the rather high affinities the mutated or noncognate tRNAs retain for the native AspRS. From these considerations, it can be expected that tRNAs favorably mischarged are those having sequence elements closely related to the tRNAAsp identity set. This is actually the case since the aspartate identity set is partially conserved in yeast tRNAAsn and tRNAGlu with a sole difference found in one anticodon position.
In this work, aspartylation of total yeast tRNA and of pure noncognate tRNA species were conducted under an aspartate concentration (500 µM) that approximates the physiological level of aspartic acid present in yeast cells (24). Noteworthily, yeast AspRS is rather unique among synthetases with a low affinity (2.5 mM) for its amino acid substrate (23, 41). Therefore, this enzyme needs a high aspartate concentration for optimal catalytic activity. Thus, at a low aspartic acid concentration (25 µM, the usual concentration in standard aspartylation assays), no significant aspartylation enhancement was observed, but mischarging was easily detected when working at a higher amino acid concentration (Fig. 3). Indeed, enhancing the amino acid concentration from 25 to 500 µM enhances by about 17-fold the specific activity of AspRS (see "Experimental Procedures"), which not only favors aminoacylation of cognate tRNAAsp but also facilitates the less efficient mischarging of noncognate tRNAs. The reason for that is the existence of an equilibrium between aminoacylation (amino acid concentration-dependent) and deacylation (amino acid concentration-independent) of tRNAs (42, 43).
The consequence of yeast AspRS mischarging ability would be dramatic misincorparations of aspartic acid residues at asparagine and glutamate positions in polypeptides. This possibility receives support considering that the high level of tRNAAsn and tRNAGlu mischarging was found under conditions converging to the physiological environment. Indeed, the high intracellular concentrations of tRNAs (~1 µM for each tRNA species, see Ref. 44) and aspartate (~500 µM aspartate, see Ref. 24) are those used in this work. However, as seen in Fig. 3, the level of tRNA mischarging can be modulated since it is dependent upon the AspRS and aspartic acid concentrations. It can even become undetectable if these concentrations are low enough.
This latent mischarging of AspRS, enhanced by the nonspecific tRNA
binding properties of its N-terminal extension, represents a vital
threat for the yeast cell. Thus, one can question whether elongator
factor would discriminate efficiently enough the mischarged tRNAs (45,
46) and protect the cell against their toxicity. Another way to lower
the risk of errors during translation would be to reduce the
intracellular concentrations of AspRS, thereby decreasing mischarging
possibilities. It is remarkable that traces of this potential
mischarging ability remain present in organisms other than yeast.
Indeed, an evolutionary proximity between tRNAAsp and
tRNAAsn has been explicitly demonstrated in some
microorganisms, with AspRS mischarging tRNAAsn and
Asp-tRNAAsn being the precursor for the synthesis of
Asn-tRNAAsn (47, 48). It is also interesting to note that
maybe evolution answered to the menace of tRNAGlu
mischarging by replacing the nature of its discriminator base in higher
eukaryotes (G73 in prokaryotes and unicellular eukaryotes changed to A
in higher eukaryotes, see Ref. 49).
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ACKNOWLEDGEMENTS |
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We thank Gilbert Eriani, Catherine Florentz, and Daniel Kern for advice and stimulating discussions and Gérard Keith, Caroline Paulus, and Anne Théobald-Dietrich for preparation of biological material.
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FOOTNOTES |
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* This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS), the Ministère de l'Education Nationale, de la Recherche et de la Technologie (MENRT), and Université Louis Pasteur, Strasbourg.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
0033-388-41-70-58; Fax: 0033-388-60-22-18; E-mail:
R.Giege@ibmc.u-strasbg.fr.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M211035200
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ABBREVIATIONS |
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The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; RS, tRNA synthetase; DTNB, 5,5'-dithiobis(nitrobenzoic acid); mcm5s2U, 5-methoxycarbonylmethyl-2-thiouridine.
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