Misacylation of tRNA with methionine in
Elizabeth Wiltrout1, Jeffrey M. Goodenbour2, Mathieu Fre ´chin3and Tao Pan2,*
1Department of Chemistry,2Department of Biochemistry and Molecular Biology, University of Chicago,
Chicago, Illinois, 60637, USA and3Institute of Molecular Life Sciences, University of Zurich, CH-8057 Zurich,
Received May 11, 2012; Revised July 30, 2012; Accepted July 31, 2012
by aminoacyl-tRNA synthetases controls transla-
tional fidelity. Although tRNA synthetases are gen-
erally highly accurate, recent results show that the
methionyl-tRNA synthetase (MetRS) is an exception.
MetRS readily misacylates non-methionyl tRNAs
at frequencies of up to 10% in mammalian cells;
such mismethionylation may serve a beneficial role
for cells to protect their own proteins against
oxidative damage. The Escherichia coli MetRS
mismethionylates two E. coli tRNA species in vitro,
and these two tRNAs contain identity elements for
mismethionylation. Here we investigate tRNA mis-
methionylation in Saccharomyces cerevisiae. tRNA
in vivo as in mammalian cells. Both cognate and
kinetics upon cycloheximide treatment. We identify
specific arginine/lysine to methionine-substituted
indicating that mismethionylated tRNAs are used
in translation. The yeast MetRS is part of a
complex containing the anchoring protein Arc1p
and the glutamyl-tRNA synthetase (GluRS). The re-
combinant Arc1p–MetRS–GluRS complex binds and
mismethionylates many tRNA species in vitro. Our
results indicate that the yeast MetRS is responsible
for extensive misacylation of non-methionyl tRNAs,
and mismethionylation also occurs in this evolution-
Translational fidelity, which is critical for cell survival,
depends on the incorporation of the correct amino
acid to its transfer RNA (tRNA). Aminoacyl-tRNA
synthetases (aaRSs) aminoacylate tRNAs with their
cognate amino acids. Despite the high fidelity of catalytic
and editing domains in aaRSs, misacylation can occur
when the aaRS catalyzes the aminoacylation of a
cognate tRNA with a non-cognate amino acid (1). aaRS
can also misacylate tRNAs through catalyzing the
aminoacylation of a non-cognate tRNA with a cognate
amino acid. Misacylated tRNAs that are used in transla-
tion produce mutant proteins. However, mistranslation at
low levels is not always detrimental to cells and in some
cases is tolerated or may even be beneficial in stress
A recent study has provided evidence that tRNA
misacylation with methionine is actively regulated in
mammalian cells and tRNA mismethionylation may
provide a benefit to cells under oxidative stress (4).
Previous findings show that genetically encoded methio-
nine residues can protect proteins against reactive oxygen
species by oxidation of methionine residues on the surface
or near active sites of proteins (5,6). Mismethionylated
tRNAs can extend this protective function by substituting
certain non-methionine residues in proteins at strategic
locations. For this function, methionine substitution
through mismethionylated tRNAs is likely to occur at
solvent-exposed residues on the surface or near the
active site of target proteins; such residues more
commonly have charged or polar side chains.
karyotes and eukaryotes. The recombinant, purified
E. coli methionyl-tRNA synthetase (MetRS) mismet-
hionylates two E. coli tRNAs in vitro, one coding for a
charged amino acid, tRNAArgCCU, and the other for a
polar amino acid, tRNAThrCGU(7). In mammalian cells,
MetRS is part of an 11 protein complex, eight of which are
aaRSs (8,9). The affinity purified human complex contain-
ing MetRS can mismethionylate two human tRNAs
coding for a charged amino acid, tRNALysCUU and
tRNALysUUU(4). In mammalian cells, mismethionylation
occurs at a basal level of ?1%, which includes tRNAs
coding for several charged amino acids. This level of
*To whom correspondence should be addressed. Tel: +773 702 4179; Fax: +773 702 0439; Email: email@example.com
Nucleic Acids Research, 2012, Vol. 40, No. 20Published online 31 August 2012
? The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
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mismethionylation increases to up to 10% under innate
immune activation and chemically triggered oxidative
Here we investigate tRNA misacylation with methio-
nine in baker’s yeast, Saccharomyces cerevisiae, both
in vivo and in vitro. Saccharomyces cerevisiae is evolution-
ary distant from mammals and bacteria, so studies of
yeast broaden the evolutionary reach of this unusual
behavior in altering the aminoacylation and translational
fidelity. Using tRNA microarrays and classical filter reten-
tion assays, we identified many mismethionylated tRNAs
in S. cerevisiae. The extent of misacylated tRNAs is de-
pendent on yeast growth conditions. Mismethionylated
tRNAs show similar in vivo utilization kinetics as correctly
charged tRNAMets, and several misacylated peptides are
detected by mass spectrometry, indicating that they are
used in translation. The yeast MetRS is part of a three
protein complex that includes a general tRNA-binding
[GluRS, (10)]. Previous reports have shown that the anti-
codon is not sufficient for tRNA binding to the yeast
requires primary, secondary and tertiary determinants in
the tRNA (11–13). The Arc1p protein binds to the MetRS
and GluRS by N-terminal interactions and to tRNA by
C-terminal interactions, which facilitate tRNA binding to
the MetRS and GluRS for aminoacylation with methio-
nine and glutamic acid, respectively (10). However, since
Arc1p binding to tRNA is non-specific, tRNA binding to
the MetRS determines the specificity of aminoacylation
(10,14). We examine tRNA binding to the recombinant
microarrays and show that AME can bind almost all
yeast tRNAs, consistent with the previous reports. We
also show that AME extensively mismethionylates many
tRNAs in vitro. Our results suggest that MetRS being a
part of a multi-protein complex provides eukaryotes with
another mechanism of tRNA mismethionylation by
allowing many tRNAs to bind to the MetRS for
misacylation with methionine.
MATERIALS AND METHODS
Yeast strains used in this study were WY798 (MAT?
URA3 LEU2 TRP1) (15), BY4742 (MAT? his3D1
leu2D0 lys2D0 ura3D0) and ?Arc1 (MATa his3D1
leu2D0 lys2D0 ura3D0 arc1D). BY4742 and ?Arc1 were
purchased from Open Biosystems. Yeast strains were
grown on YPDA medium at 30?C. Single colonies were
grown overnight in synthetic complete (SC) media. For
stationary phase experiments, these cultures were grown
to OD6004–8. For mid-log phase experiments, the over-
night cultures were diluted to OD6000.1 and grown to
35S-Met pulse labeling of yeast cells was adapted from
established procedures with minor modifications. Briefly,
yeast cells were starved for methionine by spinning down
and resuspending in an equal volume of SC–Met media
1h prior to pulse labeling to maximize35S-signal. After
pelleting, yeast cells were resuspended in 300ml of pulse
labeling media consisting of 0.02mCi/OD600
(Perkin-Elmer, Boston, MA) in SC–Met. OD600 12.5
cells were typically sufficient to yield ?100mg RNA.
Pulse labeling proceeded at 30?C for 1 or 8 min. For
chase experiments, 300ml of SC–Met supplemented with
1mg/mL fresh methionine, and 200mg/mL cycloheximide,
where appropriate, was added after the pulse period and
incubated at 30?C for 1 min. Reactions were stopped by
addition of 300ml ice cold 0.3M sodium acetate/acetic
acid buffer with 10mM EDTA, pH 4.8, and submersion
in ice, after which cells were further rinsed twice with the
same buffer. For cycloheximide treatments, 200mg/mL
cycloheximide was maintained in the acetate/EDTA
buffer solution throughout washes and lysis to maintain
For in vitro experiments, total RNA was isolated from
yeast grown to stationary phase overnight in YPDA
medium, pelleted, resuspended in 300ml 0.3 M KCl,
50mM KOAc, and transferred to a tube containing
300ml acetate-saturated phenol-CHCl3, pH 4.8 and
vortexed three times by alternating vortexing for 1min
and incubating on ice. The sample was then spun at
14000rpm for 15min at 4?C, transferred to a new tube
containing 300ml acetate-saturated phenol-CHCl3, pH
4.8, and vortexed for an additional 1min. The sample
was spun at 14000rpm for 10min at 4?C, and the
aqueous layer was transferred to a clean tube, ethanol
precipitated twice, and resuspended in 10mM Tris, pH
7.5, 1mM EDTA.
Following pulse labeling in vivo, total RNA was isolated
from yeast by transferring the sample to a clean tube con-
taining 300ml acetate-saturated phenol-CHCl3, pH 4.8,
and 1 Retsch 7mm stainless steel ball and vortexing at
room temperature for 30min. The sample was then spun
at 14000rpm for 15min at 4?C and followed the remain-
ing procedure as in the in vitro experiments. Once resus-
pended in 10mM Tris, pH 7.5, 1mM EDTA, the RNA
was again spun at 14000rpm for 15min at 4?C and
transferred to a clean tube.
Purification of the Arc1p-GluRS-MetRS complex
A plasmid overexpressing the AME complex under IPTG
control was transformed into BL21 DE3 E. coli cells. The
cells were grown in LB with 100mg/L ampicillin until
OD600 0.6, and then overexpression was induced with
0.2mM IPTG at 37?C. Expression continued for 4h,
and the cells were then harvested. Cells were lysed in
lysing buffer (50mM K-HEPES, pH 7.6, 30mM NaCl,
5mM b-mercaptoethanol) in the presence of protease in-
hibitors and 2000U DNase per 50mL extract. Following
centrifugation, the complex was purified by FPLC by
elution from a Ni-NTA column using an imidazole
gradient. The purification buffers contained 50mM
K-HEPES, pH 7.6, 150mM NaCl, 5% glycerol, 10mM
Nucleic Acids Research, 2012,Vol.40, No. 2010495
BME, and 20mM or 500mM imidazole. The complex
eluted around 300mM imidazole.
Gel filtration of the Arc1p–GluRS–MetRS complex
The affinity purified AME complex was passed through a
Superdex 200 column at 4?C to analyze by gel filtration
using the buffer containing 20mM Tris, pH 7.4, 30%
glycerol, 2mM DTT and 1M NaCl.
In vitro transcription
Saccharomyces cerevisiae tRNAMetCAU, tRNAGluCUC,
tRNAGluUUC(12) and tRNAGluUUC(1) sequences were
obtained from the genomic
Mutants 1-3 were created by swapping nucleotides from
the tRNAGluCUCand tRNAGluUUC(12). For tRNAMetCAU,
the transcript was made by in vitro transcription of
overlapping oligonucleotides and purified as described
All mature tRNAGlustart with U at the 50-position.
Since T7 RNA polymerase transcription works poorly
with U-starting RNA, the three tRNAGlutranscripts and
mutants 1-3 were first transcribed similarly as the
tRNAMettranscript but with a 50-leader sequence of
50-gggacaaata-tRNAGlu. These transcripts were then
cleaved with Bacillus subtilis RNase P holoenzyme to
obtain the appropriate tRNA sequences. The cleavage
was performed by first reconstituting the B. subtilis
RNase P holoenzyme (19). The final buffer concentration
of the reconstituted holoenzyme was 50mM Tris-HCl, pH
8, 18mM MgCl2, 0.2 M NH4Cl. The holoenzyme was
reconstituted by first mixing P RNA with water and
Tris–HCl, pH 8, and heating at 85?C for 2min, then at
room temperature for 3min, followed by adding MgCl2
and incubating at 50?C for 5min, and finally adding equal
moles of P protein and NH4Cl and incubating at 37?C for
5min. The transcription mixture was then incubated with
the reconstituted B. subtilis RNase P at 37?C for 5min.
Cleavage was stopped with 15mM EDTA, and the
cleaved transcription mixture was ethanol precipitated
and purified by denaturing PAGE.
Filter-based aminoacylation reactions
Filter-based aminoacylation reactions with methionine
were performed at 30?C in 20mM K-HEPES (pH 7.2),
100mM methionine, 10mM MgCl2, 5mM DTT, 4mM
ATP, 150mM NH4Cl, 0.1mM EDTA,
L-[35S] methionine, 0 or 2.5mM tRNA transcripts and
0.1mM AME enzyme. Filter-based aminoacylation reac-
tions with glutamic acid were performed at 30?C in
100mM K-HEPES (pH 7.2), 100mM glutamic acid,
10mM MgCl2, 10mM DTT, 2mM ATP, 30mM KCl,
50mM L-[3H] glutamic acid, 0 or 2.5mM tRNA transcripts
and 0.1mM AME. Filters were counted on a Perkins–
Elmer scintillation counter. For kinetics experiments,
aminoacylation reactions were performed with up to
In vitro aminoacylation reactions
In vitro aminoacylation of tRNA for microarray analysis
was performed at 30?C for 6 or 20min in 20mM
K-HEPES (pH 7.2), 100mM NH4Cl, 0.1mM Na-
EDTA, 2mM ATP, 1.5mM MgCl2, 2.5mM DTT,
MA), 0.4mg/mL gel-purified total yeast tRNA and
0.5mM purified AME enzyme.
L-[35S]methionine (Perkins–Elmer, Boston,
50-32P-tRNA binding to Arc1p–GluRS–MetRS complex
Total tRNA was gel-purified from total RNA isolated
from yeast at pH 4.8. The purified tRNA was dephos-
phorylated with calf intestinal phosphatase in 50mM
Tris, pH 8, 0.1mM EDTA, extracted from phenol/
50-32P-labeled with T4 PNK and renatured. Binding ex-
periments contained 10pmol AME with excess (40pmol
total) renatured tRNA doped with 50-32P-labeled tRNA in
0.1M K-HEPES, pH 7.2, 1.5mM Mg2+and 0.1–0.4M
KCl. The binding mixture was incubated at 30?C for
10min and then added to Genscript Ni2+-MagBeads fol-
lowing the Genscript protocol 2.1.2 for purification of
polyhistidine-tagged proteins under native conditions.
The wash and elution buffers were the same as used in
the Ni-NTA purification of the AME complex. The
eluted tRNA was ethanol precipitated and analyzed by
Hybridization to microarrays and controls using radio-
active detection on a Genomic Solutions Hyb4 station
has been described previously (4). Mismethionylation
and tRNA binding to AME for yeast was assessed with
manually printed arrays containing 40 nuclear and 24
mitochondrial probes for S. cerevisiae and 31 probes for
E. coli as controls. The arrays contained eight replicates
for each probe. Experiments with35S-Met detection used
20mg total RNA per array. Signals were quantified using
Fuji Imager software.
Mass spectrometry analysis
Mass spectrometry data and FASTA sequences for nine
abundant yeast proteins (ADH1, CDC19, ENO1, ENO2,
FBA1, PDC1, PGK1, TDH2 and TDH3) were obtained
from Geiler-Samerotte et al. (20) and were analyzed by
MaxQuant (21). Additional FASTA sequences were
created for each protein with one methionine substitution
at each lysine and arginine residue. As trypsin-digested
peptides are cleaved at Lys and Arg residues, the
MaxQuant data were analyzed for longer peptides repre-
senting a methionine misincorporation at the lysine and
tRNA mismethionylation in yeast cells
To determine if tRNAs are misacylated in S. cerevisiae, we
chose to work first with the S. cerevisiae strain 798, a fully
prototrophic strain whose tRNA abundance and charging
10496 Nucleic Acids Research, 2012,Vol.40, No. 20
characteristics have been characterized previously (15).
We detected tRNAs that are either correctly or incorrectly
aminoacylated with methionine after pulse labeling with
35S-Met using arrays containing probes for all cytosolic
and mitochondrial tRNAs of S. cerevisiae. The array
includes eight repeats each of 40 cytosolic and 24 mito-
chondrial S. cerevisiae tRNA probes. In addition, the
array includes eight repeats each of 1 blank control and
31E. coli tRNA probes, which serve as negative controls.
The probe sequences used were identical to those
described previously to measure tRNA charging in yeast
and E. coli (22,23).
probes for both methionyl and non-methionyl-tRNAs
(Figure 1A). The most intense signals were derived from
tRNAMets, as expected. The strongest signal was derived
from cytosolic elongator tRNAMete. Signals from mito-
chondrial tRNAs were weaker than tRNAMete, presum-
ably due to the significantly lower abundance of these
tRNAs. Unexpectedly, signal from cytosolic initiator
tRNAMeti was much weaker than that of tRNAMete,
even though the abundance and charging levels of
tRNAMetiand tRNAMeteshould be similar according to
previous studies (23,24). Varying pulse labeling time from
1 to 8 min did not markedly change this behavior (data
not shown). This result is significantly different from
mammalian studies where similar levels for35S-charging
signals were observed for tRNAMetiand tRNAMete(4).
At this time, we do not understand the reasons for the
labeling. One possible explanation is that yeast cells may
use distinct intracellular methionine pools to charge
tRNAMetiand tRNAMete; our result would be consistent
with methionine used for tRNAMetecoming from imme-
diate Met uptake and methionine used for tRNAMeti
coming from Met obtained or de novo synthesized at
We performed a series of controls to ensure that the
majority of the
tRNAsare derived from
(Figure 1B) as was done previously for the mammalian
misacylation study (4). To rule out signals due to
cross-hybridization, we added excess oligonucleotides
tRNAMetto the hybridization mixture. All signals from
Met-tRNAs could be eliminated with little change in
the signal intensity from non-Met-tRNAs. To rule out
signals due to peptidyl-tRNAs with an N-terminal
aminopeptidase-M before array hybridization. Most
peptidase treatment (Figure 1C). Post-transcriptional
thio-modifications of tRNA may be radio-labeled via
post-transcriptional thio-modifications of tRNA, we
hydrolyzed the labile aminoacyl bond of the charged
tRNA sample at pH 9 prior to array hybridization to
remove all35S-signal due to methionine charging. Yeast
tRNAs known to contain thio-modifications at the wob-
ble position of the anticodon include tRNALysUUU,
35S-signals from numerous yeast tRNA
35S-detected charging levels of tRNAMeti in pulse
35S-signals present in non-methionyl-
the RNA samplewith
35S-Met. To distinguish signals due to
tRNAGluUUC, tRNAGlnUUG (5-methoxycarbonylmethyl-
tRNAThrIGU(2-thio-U or s2U (25)). Signals from these five
tRNAs were readily detected after deacylation. Surpris-
ingly, signals from several other tRNAs were also present
tRNAThrUGU; these tRNAs do not contain known
whether these deacylation resistant
derived from thio-modifications in these tRNA.
We performed one additional control to ensure that
misacylation observed in yeast is not caused by methio-
nine starvation in the standard pulse-labeling protocol
(Figure 1D). In the standard pulse-labeling experiments,
yeast cells were first starved for methionine for 1h before
the addition of35S-Met. This step decreases the intracel-
lular pools of cold Met, resulting in increased specific
activity of Met in labeled protein or RNA. This step
was not necessary for mammalian cells because they are
intrinsically auxotrophic for Met, and the intracellular
pools of cold Met is much lower. Misacylation still
occurred when yeast had not been starved prior to pulse
labeling, although signals for both Met and non-Met-
tRNAs were substantially reduced.
A summary of the misacylation result is shown in
Figure 1C. Approximately two-thirds of all non-Met-
tRNA probes show detectable
deacylation prior to array hybridization removed signals
from ?70% of these probes, suggesting that the majority
of these signals are derived from mismethionylation. No
mismethionylation was detected for all mitochondrial
tRNAs. At the stationary phase, the cumulative extent
of misacylation is over 10% relative to all Met-tRNAMet
signals. Since the35S-signal for tRNAMetiis significantly
lower in yeast than in mammalian cells and the
misacylation extent is normalized to
Met-tRNAs, this level of mismethionylation in yeast is
comparable to the highest level observed in mammalian
We determined whether tRNA mismethionylation
depends on cell growth conditions in S. cerevisiae
(Figure 2A and B). The growth state of yeast is known
to impact numerous cellular factors, including gene
expression, metabolic rate and oxidative stress load
(26–28). In addition, the stationary phase is thought to
impose an oxidative stress relative to the mid-log
phase (29). Yeast 798 strain was grown to either mid-log
(OD600?0.5) or stationary (OD600?4–8) phase and pulse-
under both conditions; however, a greater extent of
misacylation occurred in the stationary phase than in the
mid-log phase (Figure 2B). This result indicates that the
extent of misacylation depends on the yeast growth phase.
In mammals, mismethionylation is increased upon innate
immune or chemically triggered oxidative stress.
In vitro experiments using HeLa multi-synthetase
complexes containing MetRS suggest that the higher
order structure of MetRS may play a role in regulating
misacylation (4). Yeast MetRS is part of a three protein
complex, including the GluRS and a general tRNA
binding protein Arc1p. We tested a potential role of
35S-signals are also
35S-signals of all
35S-Met. Mismethionylation was observed
Nucleic Acids Research, 2012,Vol.40, No. 2010497
+ AP pH 9
Met Met-starvation: No
Pulse time: 1’ 8’ 1’
Figure 1. tRNA misacylation with methionine in yeast cells. The full array is shown in panel A. For easier viewing of results, only three array blocks
containing the probes of Mete-tRNA and three examples of the misacylated tRNAs, Ala-IGC, Ala-UGC, and Asp-GUC, are shown in panels B and
D and in Figures 2 and 3. (A) RNA from the35S-Met pulse-labeled stationary phase S. cerevisiae strain 798 was hybridized to a microarray showing
many potentially misacylated tRNAs. Strain 798 is a fully prototrophic strain. All Met-tRNA probes (two for cytosolic and two for mitochondrial)
are shown as black squares in the array layout. (B) Array controls for mismethionylation include cross-hybridization with excess free Met-tRNA
probes (+Met probes), peptidyl-tRNA following treatment with aminopeptidase M (+AP), and thio-modification following deacylation (pH 9
deacylation). In this selected array view, the four strong spots remaining correspond to tRNAArgUCUwhose mouse homolog contains a known
thio-modification. On the array layout, black=Met-e; green=non-Met yeast tRNAs; cyan=yeast tRNAArgUCU. (C) Semi-quantification of
misacylation results with and without aminopeptidase treatment. Many cytosolic tRNAs are misacylated, but no misacylation for mitochondrial
tRNA was observed. Signals from the deacylation-resistant tRNAs are shown in cyan on the left. At least three of these contain known
thio-modifications. (D) Misacylation is not exclusively caused by the initial Met starvation in the standard pulse-labeling protocol.35S-Met pulse
labeling of unstarved cells results in much lower signals, but35S-Met labeling of non-tRNAMets is still detectable.
10498 Nucleic Acids Research, 2012,Vol.40, No. 20
Arc1p in tRNA mismethionylation by performing pulse-
labeling experiments with an Arc1 null yeast strain. For
this purpose, we switched to yeast cells in the BY4742
background, where the wild-type and the isogenic ?Arc1
strains are readily available. No difference in misacylation
relative to the wild-type BY4742 strain was detected
(Figure 2C). This result suggests that the MetRS alone
may be able to methionylate non-methionyl tRNAs
Aminoacylation of tRNA does not guarantee its use in
translation. At least in bacteria, the utilization of an
aminoacyl-tRNA depends upon a compromise of specific
tRNA and their charged amino acid interactions with the
elongation factor and on the ribosome (30,31). Very little
is known how yeast elongation factor (eIF1a) and yeast
ribosome choose how misacylated tRNA is utilized in
translation. We performed one experiment using cold
chase and cycloheximide to test whether mismethionylated
tRNAs are likely used in translation in yeast (Figure 3).
Cycloheximide inhibits ribosome elongation and is widely
used in cellular studies of protein synthesis. To examine
the kinetics of turnover of mismethionylated tRNAs, cells
were first pulse-labeled with35S-Met, followed by a rapid
cold chase of a large excess of non-radioactive Met in the
absence and presence of cycloheximide. The resulting
35S-labeled tRNAs were then examined by microarrays.
In the absence of cycloheximide, signals from both Met-
tRNAs and non-Met-tRNAs were reduced by ?40-fold,
suggesting that both types of charged tRNAs are turned
over with similar kinetics in cells. In the presence of
cycloheximide, the amount of35S-labeled tRNAs for the
798 strain is reduced by <1.8-fold for Met-tRNAs and
non-Met-tRNAs (Figure 3C). This result shows that in-
hibition of translation also inhibits the turnover kinetics of
mismethionylated tRNAs to a similar extent as the
turnover of correctly charged Met-tRNAs, consistent
with mismethionylated tRNAs being used in translation
in yeast cells.
To determine that misacylated tRNAs are indeed
used in translation in yeast cells, we analyzed mass spec-
trometry data from Geiler-Samorette et al. (20) using
MaxQuant (21). We chose to analyze the peptides for
some of the most abundant yeast proteins, including
nine proteins involved in glycolysis and fermentation
(Figure 4A). The proteins had been trypsin-digested,
which cleaves the peptides at Lys and Arg residues,
prior to mass spec analysis. Since lysyl- and argininyl-
tRNAs are two of the misacylated tRNA species, we
chose to look for longer peptides representing methionine
misincorporation at Lys and Arg residues. We found low
abundant peptides representing misincorporation of me-
thionine at both Lys and Arg residues in seven of these
nine proteins at a frequency of 0.66% of all observed
peptides for these proteins. Example spectra are shown
in Figure 4B for a wild-type and its mistranslated
peptide from pyruvate kinase, CDC19. The Arg codon
at this misincorporated position in CDC19 is AGA.
AGA is read by tRNAArgUCU, which is the only
tRNAArgisoacceptor that shows high levels of misacy-
lation (Figure1C). These results indicate that the misacy-
lated tRNA species are used in translation.
tRNA mismethionylation with purified yeast
components in vitro
We used recombinant, purified yeast AME complex
from E. coli to demonstrate that this complex is sufficient
to mismethionylate yeast tRNAs (Figure 5). In vitro
aminoacylation with S. cerevisiae tRNAMetpreviously
used yeast MetRS alone in the presence or absence of
Arc1p, not the full AME complex (10,32). To better
recapitulate cellular conditions, we used the AME
complex for all of our in vitro aminoacylation studies.
The reaction scheme involves incubating the purified
AME complex with total yeast tRNA with
followed by hybridization of the reaction mixture on the
microarray (Figure 5A). Our affinity purified AME
complex was derived from an overexpression plasmid in
E. coli; it contains an amino-terminal His6 tag on the
Arc1p protein. Since the His6tag is only on the Arc1p
protein, excess Arc1p appears to be purified with the
Growth: ml st
Figure 2. Growth condition dependence of tRNA mismethionylation.
(A) Signalsfrom mid-logor
strain 798 show increased mismethionylation at the stationary phase.
(B) Semi-quantitative comparison. The total amount of mismethi-
(C) Mismethionylation does not require the generic tRNA binding
protein Arc1p in the AME complex. Both BY4742 and its isogenic
?Arc1 strain show similar extent of mismethionylation.
stationary phase cellsfrom
Nucleic Acids Research, 2012,Vol.40, No. 2010499
complex during Ni-NTA affinity purification, as seen
by SDS-PAGE (Figure 5B). As expected, essentially
all synthetase molecules are associated with Arc1p, and
the majority of both synthetases form a single peak in
size exclusion chromatography (Figure 5C).
We charged total RNA isolated from stationary phase
(Figure 5D). Many tRNA species were misacylated and
mis-methionylation increases over time (Figure 5E). All
misacylated in vivo. The greater extent of misacylation
in vivo may be due to the AME complex association
with polysomes, although this remains to be determined.
The mammalian multi-synthetase complex is associated
complex alone (4). When the in vitro charging sample
was first deacylated at pH 9 followed by array hybridiza-
tion, no deacylation-resistant tRNA was observed, as
expected due to the lack of post-translational modification
enzymes in the in vitro charging mixture (Figure 5E).
Quantification of the in vitro misacylated tRNA spe-
cies showed high levels for tRNA species mostly coding
for charged and polar amino acid side chains (Figure 5D).
Although a very extensive pattern of in vitro misacylation
is present, still more mismethionylated tRNA species are
observed in vivo than in vitro. Similar results have been
The Arc1p in the AME complex is a generic tRNA-
binding protein and assists tRNA binding to the MetRS
35S-Met using the purified AME complex
and GluRS for aminoacylation (10,14). The extensive level
of misacylated tRNA species in S. cerevisiae suggests that
Arc1p may also shuttle non-Met-tRNAs to the MetRS to
be aminoacylated with methionine. We performed an
experiment to compare tRNA binding versus mis-
methionylation by the AME complex (Figure 6). We
first incubated 50-32P-labeled total yeast tRNA with the
purified AME complex at varying concentrations of KCl
followed by affinity pull down of the AME complex.
Bound32P-labeled tRNAs were then examined by micro-
array (Figure 6A and B). Almost all detectable tRNA
species are bound by AME at 0.1M KCl; increasing
KCl to 0.4M reduced the number of tRNA species
bound to the complex as expected (Figure 6C). This
result is consistent with the ability of AME to bind essen-
tially any tRNA (32). Among the bound tRNAs at 0.1M
KCl, only a subset is mismethionylated, suggesting that
mis-methionylation has additional requirement beyond
Finally, we applied the classical filter-based aminoacy-
lation assays to confirm that the AME complex is capable
of misacylating unmodified tRNA transcripts at high effi-
ciency (Figure 7). We chose to work with transcripts of
tRNAMete (Figure 7A, left) and variants of tRNAGlu
(Figure 7A, middle) because the AME contains both
tRNAGluUUCisoacceptor family has two isodecoders, a
major form with 12 copies and a minor form with a
tRNAGluUUC(1) isodecoders differ by one nucleotide in
Figure 3. Mismethionylated tRNAs have similar cellular utilization kinetics as tRNAMets. Yeast strains 798 (A) or BY4742 (B) were pulse-labeled
with35S-Met for 1min followed by 1min cold chase with a large excess of Met with or without cycloheximide. We used two strains to indicate that
the cellular utilization kinetics of mismethionylated tRNAs are not strain-specific. Strain 798 is fully prototrophic, whereas BY4742 is the parent
strain from where all isogenic deletion strains are derived. (C) Quantitative comparison of the disappearance of Met-tRNA and non-Met-tRNA
signals in the absence of cycloheximide or the maintenance of Met-tRNA and non-Met-tRNA signals in the presence of cycloheximide in strain 798.
10500Nucleic Acids Research, 2012,Vol.40, No. 20
their acceptor stems, and the isoacceptor tRNAGluCUC
differs by five nucleotides from tRNAGluUUC(12). These
three tRNAGlus were not distinguishable on our micro-
arrays due to their sequence similarities. We also made
mutants (Figure 7A, right) in the
acceptor stems and anticodon loop to probe possible
sequence identity elements of misacylation.
The purified AME complex charged all the tRNA tran-
scripts with their cognate amino acids, tRNAMete with
(Figure 7B). All three tRNAGluvariants as well as all
similar levels under this reaction condition. As a positive
control, very little mischarging of tRNAMetewith Glu is
present, showing that misacylation occurs exclusively by
the MetRS (Figure 7B). This result suggests that all three
tRNAGluvariants could have contributed to the total
35S-signal for the tRNAGluon our microarray. We also
measured the charging kinetics of tRNAMete and the
major form of tRNAGluwith Met or Glu (Figure 7C). A
two-phase charging kinetics was observed in all cases.
Both phases have essentially the same Km values, but
they differ by over 100-fold in kcatvalues. The reason
for such two-phase charging behavior by the purified
AME complex is unclear. However, both the Kmand the
kcatvalues for the fast phase is within the same order of
magnitude observed previously, although all previous
experiments were performed with just the MetRS or
GluRS protein not the AME complex. The AME
charged tRNAMetewith Met at kcat/Kmof 0.39mM?1s?1
and tRNAGluUUC(12)with Glu at kcat/Kmof 1.1mM?1s?1,
indicating that our recombinant AME is highly active.
The AME mischarged tRNAGluUUC(12)with Met at kcat/
Km of 0.2mM?1s?1, which is only 2-fold lower than
cognate charging. This result is significantly different
from in vitro mischarging of the AME complex using
total yeast tRNA (Figure 5D), which suggests that
certain modifications in tRNAGlu
We have demonstrated here that tRNA misacylation with
methionine occurs in S. cerevisiae. This represents the
second example of tRNA mismethionylation in cells in
Figure 4. Mismethionylated tRNAs are used in translation. (A) Glycolysis pathway showing the nine proteins analyzed in this study from a
proteomic mass spectrometry dataset. These nine proteins were chosen based on their high occurrence in this mass spec dataset. For simplicity,
only Lys/Arg!Met substitutions were searched for in our analysis. Thirty-one peptides from a total of 4675 peptides identified are derived from
K/R!M substitutions (0.66%). Proteins with (maroon) or without (blue) detected mistranslated peptides are shown. (B) Spectra for one example of
a mistranslated peptide (top) and its wild-type peptide (bottom) in CDC19, the pyruvate kinase. The AGA codon at this mistranslated position
should be read by tRNAArgUCU, which is the only tRNAArgisoacceptor misacylated with Met. As expected, the relative abundance of the
mistranslated peptide is significantly lower compared to the wild-type peptide (Y-axis scale on the right).
Nucleic Acids Research, 2012,Vol.40, No. 2010501
evolutionarily distant from mammals, this result suggests
that tRNA mismethionylation is conserved from fungi to
mismethionylation is derived from the activity of the
MetRS. Mismethionylation occurs in vitro when the
yeast MetRS is associated with the other two proteins in
the AME complex. This association in the complex,
however, is not required for mismethionylation in vivo as
an Arc1 deletion yeast strain also shows similar level of
mismethionylation. We further show by in vivo utilization
misacylated tRNAs are used in translation in yeast, as in
mass specanalysis that
Our results lead to two wide-open biological questions.
First, how do ribosome choose mismethionylated tRNAs
in translation? Misacylated tRNA may or may not be used
in translation depending on elongation factor selection
and ribosome utilization. In E. coli, misacylated tRNAs
can bind to the elongation factor EF-Tu at different
affinities compared to correctly charged tRNAs (30,35).
This differential binding has been shown to result in the
exclusion of some misacylated tRNAs to EF-Tu binding
while some other misacylated tRNAs cannot be properly
delivered into the A-site of the ribosome (31). How
mismethionylated tRNAs are selected by EF-Tu is,
however, unclear as the EF-Tu selection of misacylated
Figure 5. Mismethionylation of yeast tRNAs by recombinant AME in vitro. (A) Experimental flow. (B) SDS-PAGE analysis of purified recombinant
AME from E. coli. The complex is His6-tagged at Arc1p, which explains a possible over-representation of the Arc1p protein in this preparation. (C)
Size exclusion chromatography using a gel filtration Superdex 200 column shows that the AME complex contains both synthetases associated with
Arc1p. (D) Total, deacylated tRNA isolated from stationary phase yeast strain 798 was gel purified and then charged with35S-Met using purified
AME. The RNA from 20 min charging was hybridized to the array in the presence of excess free tRNAMetprobes. The array layout shows cytosolic
Met-tRNA probes as black squares and non-Met-tRNA probes showing misacylation as green squares. Following 20min charging, another sample
was deacylated and hybridized to the array. The deacylation resistant signals from the cellular yeast samples are absent from the array as expected.
The remaining signal is derived from incompletely deacylated tRNAMete. (E) Quantification of in vitro results showing more misacylation occurs after
10502Nucleic Acids Research, 2012,Vol.40, No. 20
tRNAs depends on the tRNA and the amino acid identity;
mismethionylated tRNAs were not used in previous
studies. In the fungal CTG clade species, the tRNA with
anticodon CAG is charged with either serine or leucine,
and both Ser and Leu charged tRNAs are used in trans-
lation (36). This is one example of experimental evidence
for the utilization of mischarged tRNAs in eukaryotes,
suggesting that EF-1a does not vigorously discriminate
misacylated tRNAs. Many studies have been conducted
mismatched tRNAs in the A and the P sites. Misme-
thionylated tRNAs, however, can enter the A site while
maintaining perfect codon–anticodon
instance, a mismethionylated tRNALysCUUis expected to
enter the A site containing the cognate AAG codon. We
do not know how mismethionylated tRNA in the A site
might perturb peptide bond formation or translocation,
due to a lack of previous studies that specifically considers
Although EF-1a may not discriminate misacylated
tRNAs, our proteomic analysis here shows that misacy-
lated lysyl- and argininyl-tRNAs are used in translation.
mismethionylated tRNAs in the future, it should be
possible to conduct proteomic studies to specifically
identify Met substitutions in proteins that can be con-
sidered to derive from all the mismethionylated tRNA
species. Each mismethionylated tRNA species is only
present at an average level of ?0.5% of tRNAMets. If
we consider that ribosome uses all such tRNAs, it will
still represent a sub-1% presence of Met at individual
non-Met positions in proteins. Met-containing peptides
are also prone to oxidation in mass spectrometry
peptides, which can be specifically enriched from the
proteomic mixture, no reliable chemical method is yet
available to enrich Met-containing peptides.
The second biological question deals with the potential
tRNAs. We have proposed previously for mammalian
cells that low-frequency substitution of non-Met residues
with Met in stress response proteins can enhance the
identify the rulesofribosomeutilizationof
Figure 6. tRNA binding to the AME complex in vitro. (A) Experimental flow. (B) Purified yeast tRNA was 50-32P-labeled and incubated with AME
at varying concentrations of KCl. Arrays show the total tRNA input and tRNAs eluted from AME in the presence of 0.1M KCl or 0.4M KCl. The
array layout shows all cytosolic tRNA probes as black squares and all mitochondrial tRNA probes as light gray squares. (C) Comparing in vitro
misacylation to tRNA binding to AME. All mismethionylated tRNAs in vitro bind to AME at 0.1M KCl.
Nucleic Acids Research, 2012,Vol.40, No. 2010503
Met residues against ROS inactivation of cells’ own
proteins. ROS refers to a collection of highly reactive
radicals or peroxides, byproducts of the electron transport
chain, and as such used as signaling molecules for cell
health and stress (37). In mammals, ROS is also used as
chemical weapons against invading microbes or undesired
molecules. Their high chemical reactivity easily leads to
damages of a cell’s own molecules, including proteins.
To protect their own proteins against ROS inactivation,
certain Met residues in an endogenous protein are pos-
itioned at strategic places to react first with diffusing
ROS molecules before they can oxidize sensitive amino
acid side chains in, e.g. an active site of an enzyme to
result in permanent inactivation (5,38). Our previous
proposal suggests that substituting certain non-Met
residues with Met during translation can enhance this
effect, in particular, during oxidative stress response.
Our yeast result here is consistent with this idea in that
many tRNA encoding charged and polar amino acids are
mismethionylated so that a Met substitution at these
residues would enable such a protective function but less
likely produce misfolded proteins. In order to test
mutants that have diminished ability to mismethionylate
but still maintain a wild-type level activity to charge
tRNAMet. Such MetRS mutants are available for the E.
coli enzyme (7), and we are actively identifying such
mutants using the recombinant AME as the starting point.
To rule out misacylation caused by other enzymes in
yeast cells, we purified the recombinant AME complex
from E. coli and performed in vitro aminoacylation reac-
tions with purified total yeast tRNA and tRNA tran-
scripts. Mismethionylation was prevalent for many
tRNAs and for all tRNAGlutranscripts tested. All the
misacylated tRNAs in vitro are also misacylated in vivo,
suggesting that the AME complex binds many tRNAs and
facilitates interaction of many tRNAs with the MetRS for
methionylation. It appears that the Arc1p protein in
the AME complex facilitates interactions of MetRS with
tRNAMetand a large number of other tRNAs. However,
Arc1p is not required for methionylation in vivo.
Figure 7. Mismethionylation of tRNAGlutranscripts. All tRNAGlutranscripts were prepared by first making T7 transcripts containing a 50-leader.
The 50-leader was then removed by RNase P cleavage to produce mature tRNAGlucontaining U+1. (A) Sequence of the mature tRNAMeteand
tRNAGlutranscripts. Left: tRNAMete; middle: tRNAGlufrom the reference yeast genome; right: tRNAGlumutants derived from the major
tRNAGluUUC(12). (B) Relative saturation charging after 20min of tRNAMeteand tRNAGlutranscripts with Met or Glu using the purified AME.
All tRNAGlutranscripts and mutants were mischarged with Met. However, tRNAMetwas not mischarged with Glu. (C) Km, kcatand kcat/Kmvalues
for the fast phase of tRNAMetcharging with Met (Met-tRNAMete), tRNAGlucharging with Met (Met-tRNAGluUUC) and tRNAGlucharging with Glu
10504 Nucleic Acids Research, 2012,Vol.40, No. 20
The E. coli MetRS misacylates only two E. coli tRNAs,
tRNAArgCCU and tRNAThrCGU in vitro (7). These two
tRNAs have anticodons that differ from the anticodon
for methionine, CAU, by one nucleotide. The anticodon
and several regions of the E. coli MetRS that interact with
the anticodon loop are responsible for misacylation. In
addition, several studies have shown that the CAU anti-
codon plays a key role in yeast tRNA methionylation and
discrimination (39). As there are a large number of differ-
ent misacylated tRNAs in S. cerevisiae, the misacylated
tRNAs have little similarity among their anticodons.
This raised the question of how so many different
tRNAs are misacylated with methionine in S. cerevisiae.
We found here that many tRNA species bind to the AME
complex, including all the tRNAs that are misacylated.
Since Arc1p is a generic tRNA-binding protein, Arc1p
can bind many tRNAs and likely transfer them to the
MetRS for aminoacylation with methionine, potentially
explaining why the AME complex misacylates many
tRNA species in vitro.
In summary, tRNA mismethionylation occurs in
mammals and as shown here in yeast. As in mammals, a
large number of tRNAs can be mismethionylated in yeast,
and these misacylated tRNAs are used in translation. The
misacylated tRNAs code for charged or polar amino
acids, corroborating a role for Met substituting more
provides another role of multisynthetase complexes in eu-
karyotes and expands our knowledge on the mystery of
molecular and biological roles of mis-translation.
We thank Dr Chuan He for his advisory role on EW and
his insightful comments on this project. We also thank
Dr Hubert Becker for comments on the manuscript and
Drummond for providing the mass spectrometry dataset,
Dr Marc Parisien for his computational assistance in the
mass spectrometry analysis, the members of Dr Joseph
Piccirilli’s lab for use of their scintillation counter and
Dr Thomas Jones for useful discussions.
National Institutes of Health (NIH) [DP1GM105386 to
T.P.]. An NIH pre-doctoral training grant in Chemistry
and Biology [T32-GM008720 to E.W.]. Funding for open
access charge: NIH [DP1GM 105386].
Conflict of interest statement. None declared.
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