From the Laboratoire de Biochimie Pharmaceutique, Faculté de Pharmacie, Université de Rennes I, UPRES Jeune Equipe 2311, 2 avenue du Professeur Léon Bernard, 35043 Rennes, France
Received for publication, December 17, 2002, and in revised form, February 13, 2003
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ABSTRACT |
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Transfer messenger RNA (tmRNA) directs
the modification of proteins of which the biosynthesis has stalled or
has been interrupted. Here, we report that aminoglycosides can
interfere with this quality control system in bacteria, termed
trans-translation. Neomycin B is the strongest inhibitor of
tmRNA aminoacylation with alanine (Ki value of
~35 µM), an essential step during
trans-translation. The binding sites of neomycin B do not
overlap with the identity determinants for alanylation, but the
aminoglycoside perturbs the conformation of the acceptor stem that
contains the aminoacylation signals. Aminoglycosides reduce the
conformational freedom of the transfer RNA-like domain of tmRNA.
Additional contacts between aminoglycosides and tmRNA are within
the tag reading frame, probably also disturbing reprogramming of the
stalled ribosomes prior protein tagging. Aminoglycosides impair
tmRNA aminoacylation in the presence of all of the transfer RNAs
from Escherichia coli, small protein B, and elongation
factor Tu, but when both proteins are present, the inhibition constant
is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA
chaperone activities, ensuring that tmRNA adopts an optimal
conformation during aminoacylation.
In bacteria, transfer messenger RNA
(tmRNA)1 known
alternatively as SsrA RNA or 10Sa RNA, rescues stalled ribosomes and
contribute to the degradation of incompletely synthesized peptides (for
reviews, see Refs. 1-3). In a process termed
trans-translation, tmRNA acts first as a tRNA, being
aminoacylated at its 3' end with alanine by alanyl-tRNA synthetase
(AlaRS) (4, 5) and adding an alanine to the stalled polypeptide chain.
Resumption of translation ensues not on the mRNA on which the
ribosomes were stalled but at an internal position in tmRNA.
Translation termination occurs and permits ribosome recycling.
trans-Translation plays at least two physiological roles in
bacteria: removing ribosomes stalled upon mRNAs and tagging the
resulting truncated proteins for degradation.
Because tmRNA is unique to prokaryotes and is required for
viability of some bacteria, it has attracted the attention of those interested in novel targets for antibiotic therapy. tmRNA has to be
aminoacylated before directing the addition of a peptide tag to the
problematic protein. Moreover, in Neisseria gonorrhoea, and
probably also in other bacteria, tmRNA aminoacylation is essential for resolving stalled translation complexes and preventing depletion of
free ribosomes (6), whereas tagging for proteolysis is dispensable. tmRNA aminoacylation is therefore an attractive target for blocking trans-translation in the pathogenic bacteria responsible for
infectious diseases.
The interactions of aminoglycosides with RNAs represent a paradigm in
the use of small molecules as effectors of RNA function. Aminoglycosides bind and modulate the function of a variety of therapeutically useful RNA targets (for a review, see Ref. 7) and show
antimicrobial as well as antiviral activities. Aminoglycoside antibiotics disrupt the bacterial membrane and induce miscoding during
prokaryotic protein synthesis by binding to the ribosomal A site (8).
They also interfere with translational control (9) and with human
immunodeficiency virus replication (10) and inhibit the activity of
several catalytic RNAs by displacing essential metal ions, such as
self-splicing group I introns (11, 12), hammerhead (13), human
hepatitis delta virus (14), and hairpin ribozymes (15), as well
as the tRNA processing activity of RNase P RNA (16).
Recent structural (17) and functional (18) evidence indicates that
aminoglycosides can bind and inhibit the aminoacylation of two
canonical tRNAs: Escherichia coli tRNAPhe and
yeast tRNAAsp. The crystal structure of yeast
tRNAPhe in complex with neomycin B reveals that the
aminoglycoside binds in the deep groove below the D-loop.
Inhibition of aminoacylation of tRNAs is proposed to be either because
the aminoglycoside interferes with the interaction between the
aminoacyl-tRNA synthetase and its cognate tRNA through its binding to
major recognition elements (the phenylalanine system) (17) or via a
conformational change of the RNA (the acid aspartic system) (18). For
the alanine system, aminoacylation is determined by a single
G3·U70 pair (19) and also by minor identity
elements including the discriminator base A73 and a
G2·C71 pair. This limited set of nucleotides
(see Fig. 1, A and B, circled nucleotides) is conserved in all of the 293 tmRNA genes that
have been sequenced (20). tmRNA is a partial structural mimic of canonical tRNAs thanks to the presence of an acceptor stem, a T-stem
loop, and a D-analog (21). tmRNA is, however, ~5-fold bigger
compared with canonical tRNAs, contains several pseudoknots and an
internal open reading frame, probably lacks most of the key tertiary
interactions present in tRNAs, and does not contain an anticodon
stem-loop. Here, we report that several aminoglycosides interact with
tmRNA with affinity and specificity, preventing its aminoacylation
with alanine. The tRNA-like domain of tmRNA is altered upon
aminoglycoside binding. Chemical footprints in solution have explored
the structural basis of the interaction between tmRNA and several
aminoglycosides. trans-Translation is therefore a novel
target for aminoglycosides.
Enzymes and RNAs--
Alkaline phosphatase and T4 polynucleotide
kinase are from New England Biolabs
(Beverly, MA). T4 RNA ligase was from
Invitrogen. RNases S1, V1, U2, and
T1 were from Amersham Biosciences.
[ Aminoacylation Experiments--
Recombinant alanyl-tRNA
synthetase was purified on Ni2+-nitrilotriacetic
acid-agarose (Qiagen), and purity was confirmed on a 10% SDS-PAGE.
Five independent protein purifications were required. The final protein
concentration ranged from 0.5 to 3.5 µM. The assays were
performed at 20 or 37 °C in a medium containing 50 mM
Tris-HCl (pH 7.5), 10 mM KCl, 20 mM
Ultraviolet Absorbance Melting Curves--
The progressive
melting of tmRNA and tmRNA-TLD was monitored by following their
UV absorbency at 258 nm as a function of temperature on a
UVIKONXL (Bio-tek Instruments) equipped with a temperature
regulator and with a six-cell holder. The temperature was increased
gradually at 0.5 °C/min from 15 to 93 °C. The measurements were
done in 20 mM potassium phosphate (pH 5.8), 0.5 mM EDTA, and 5 mM MgCl2 for
tmRNA-TLD and in 20 mM potassium phosphate (pH 5.8), 50 mM NaCl, and 5 mM MgCl2 for
tmRNA in the absence and presence of 500 µM neomycin
B, 10 mM tobramycin, and 2 mM paromomycin.
Chemical Footprints--
Labeling at the 5' ends of tmRNA
and tmRNA-TLD were performed with [ Inhibition of tmRNA Aminoacylation by Aminoglycoside
Antibiotics--
Aminoacylation experiments with purified
E. coli alanyl-tRNA synthetase were performed on two RNAs
purified in vivo: tmRNA from E. coli (363 nucleotides; Fig. 1A) and
tmRNA-TLD (61 nucleotides; Fig. 1B), a shorter RNA
recapitulating the tRNA-like domain of tmRNA. tmRNA-TLD is
capable of being aminoacylated with alanine in
vitro.2 The structures of the six aminoglycosides
tested are shown in Fig. 1C. Aminoglycosides are subdivided
into three major families (23), and representatives of each family were
tested. Paromomycin and neomycin B belong to the neomycin family;
amikacin, tobramycin, and kanamycin A are members of the kanamycin
family; and gentamicin represents the gentamicin family. All six
aminoglycosides inhibit aminoacylation of tmRNA with alanine, in a
concentration-dependent manner (Fig.
2). In the presence of each
aminoglycoside, the decrease of aminoacylation is not a consequence of
tmRNA or tmRNA-TLD degradation (not shown). Neomycin B is the
most efficient inhibitor, with apparent inhibition constants
(Ki) of ~35 µM for tmRNA-TLD and
~70 µM for tmRNA (Fig. 2 and Table
I). Neomycin B inhibits the
aminoacylation of tmRNA-TLD with alanine at a ~30-fold lower concentration than that required to inhibit tRNAAla
aminoacylation (Table I). Paromomycin, gentamicin, and amikacin are
also potent inhibitors, whereas kanamycin A and tobramycin are modest
ones, with a substantial difference in activity between the two RNAs
(kanamycin A and tobramycin have no measurable Ki on
tmRNA-TLD alanylation). Compared with tmRNA, aminoacylation of
tmRNA-TLD is half-inhibited at ~2-fold higher concentrations of
paromomycin, gentamicin, and amikacin (the opposite effect is observed
for neomycin B). Aminoacylation with alanine of a tRNAAla
transcript can also be altered by these aminoglycosides, but the
inhibition constants are 3-15-fold higher than that of tmRNA. Amikacin, kanamycin A, and tobramycin have related structures (Fig.
1C), although different inhibition constants (Table I), especially between amikacin and the two others. Between amikacin and
tobramycin, the only differences are at positions R1 and R2 (Fig.
1C). Therefore, the chemical groups substituted at positions R1 and/or R2 of amikacin are responsible for its lower
Ki. Replacing a hydroxyl group (paromomycin) by an
amino group (neomycin B) decreases the Ki for
tmRNA 3-fold (Table I), indicating that a minor modification on the
chemical structure of an aminoglycoside can significantly increase its
inhibitory activity on tmRNA aminoacylation. All of the subsequent
functional and structural investigations were conducted using three
representatives, neomycin B, and paromomycin as potent inhibitors and
tobramycin as a modest inhibitor of tmRNA aminoacylation.
The number of charged amino groups is important for inhibition of RNA
function by aminoglycosides. Neomycin B has six protonatable amino
groups; their corresponding pKa values are indicated in Fig. 1C. In most cases, inhibition of RNA function by
aminoglycosides depends on the number of charged amino groups (24).
tmRNA-TLD inhibition by neomycin B (at its Ki of
50 µM) was monitored at five different pH levels (5.5, 6.5, 7.5, 8.5, and 9.5), centered on pH 7.5, which was used in our
assays (not shown). At pH 7.5, neomycin B inhibits 50% of
aminoacylation; when pH increases up to 9.5, neomycin B has no
inhibitory effect on aminoacylation (the amino groups are mostly
deprotonated). At pH 8.5, 70% of aminoacylation is detected; when pH
decreases to 6.5 and 5.5, inhibition of aminoacylation by neomycin B is
more efficient, leaving only 35 and 20% residual aminoacylation,
respectively. Therefore, inhibition of tmRNA aminoacylation by
neomycin B depends on the number of charged amino groups and suggests
that complex formation between tmRNA and neomycin B is mainly
driven by electrostatic interactions.
ATP is required during the first step of all the aminoacylation
reactions to form the activated aminoacyl-adenylate (in our case an
alanyl-adenylate). Therefore, aminoglycosides could bind specifically
to the catalytic site of E. coli AlaRS as structural analogs
of ATP. To test this hypothesis, increasing concentrations of ATP from
1 to 64 mM were added in the aminoacylation reaction of
tmRNA-TLD with and without 500 µM paromomycin and in
the aminoacylation reaction of tmRNA with or without 2 mM of tobramycin ([MgCl2] is kept constant at
20 mM). Increasing the concentration of ATP does not
relieve the inhibitory effect caused by the two aminoglycosides on the
aminoacylation of both RNAs (not shown). Thus, neither paromomycin nor
tobramycin function as structural analogs of ATP in the aminoacylation
reaction of tmRNA-TLD and tmRNA with alanine, respectively.
The Michaelis-Menten parameters of tmRNA-TLD and tmRNA
aminoacylation with alanine in the absence and presence of neomycin B,
paromomycin, or tobramycin have been determined (Table
II). In the absence of aminoglycosides,
the Km for tmRNA-TLD and tmRNA are
essentially the same (5-6 µM), but the
Vmax for tmRNA aminoacylation is 1 order of
magnitude lower than that of tmRNA-TLD (Table II). Compared with
tmRNA, the smaller size of tmRNA-TLD may allow a faster
turnover of the substrate during the aminoacylation reaction. For both
RNAs, gradually increasing the concentration of either neomycin B (from
15 to 60 µM for tmRNA-TLD), paromomycin (from 50 to
500 µM for tmRNA and from 100 to 1000 µM for tmRNA-TLD), or tobramycin (from 1 to 3 mM for tmRNA and from 3 to 10 mM for
tmRNA-TLD) increases the Km up to 30-fold (Table
II), but the Vmax also increases (not shown).
Alanylation is not completely inhibited at saturating antibiotic
concentration; there is significant residual aminoacylation level at
antibiotic saturation with tmRNA-TLD and in the presence of
amikacin and kanamycin A with tmRNA (Fig. 2). Thus, the inhibition
of tmRNA aminoacylation by aminoglycosides is neither competitive
nor non competitive; therefore the Ki values could
not be measured.
Inhibition of tmRNA Aminoacylation in the Presence of All tRNAs
from E. coli--
We tested whether aminoglycosides can inhibit
tmRNA aminoacylation in the presence of all 46 tRNAs from E. coli (Table III). For this
experiment, we selected paromomycin at a concentration corresponding to
its apparent inhibition constant for tmRNA alanylation (Table I).
Native tRNAAla isoacceptors represent ~6% of the total
tRNA population in E. coli (25). In all of the tRNAs from
E. coli, up to 60% of the tRNAAla isoacceptors
are able to charge alanine (100% of charging is not observed probably
because our calculated number of tRNAAla present in the
mixture is overestimated; alternatively, a fraction of
tRNAAla might be degraded). In the presence of all tRNAs
from E. coli, there is no inhibition on the aminoacylation
of the tRNA alanine isoacceptors (Table III). This result is consistent
with the fact that purified tRNAAla UGC transcript has a
~4-fold higher Ki for paromomycin, compared with
tmRNA (Table I). When tmRNA is mixed with all of the tRNAs from
E. coli, paromomycin decreases the aminoacylation level,
even in the presence of a 10-fold excess of total tRNAs (Table III).
Because paromomycin at that concentration has no effect on the
alanylation of the tRNAs, the decrease in tmRNA aminoacylation with
alanine is responsible for the overall decrease in the charging levels
of the RNA mixture containing both tRNAs and tmRNA. Thus, paromomycin can inhibit tmRNA alanylation in the presence of all of
the tRNAs from E. coli.
Magnesium Ions Cannot Reverse the Inhibitory Effect Caused by the
Aminoglycosides--
Positively charged ammonium groups of
aminoglycosides match negatively charged metal ion-binding pockets in
RNA three-dimensional structures, displacing divalent metal ions (26).
Recent structural (17) and functional (18) data indicate that the rule
also applies for specific interactions between canonical tRNAs and aminoglycosides. If true for tmRNA, inhibition of tmRNA
aminoacylation by aminoglycosides might be overcome by increasing the
magnesium concentration. The [Mg2+]/[ATP] ratio was
kept constant at 5, as in the previous aminoacylation assays, because
it affects the specificity and efficiency of the aminoacylation
reaction (27). The magnesium ion
concentration was gradually increased, and the aminoacylation of
tmRNA-TLD and tmRNA, with and without paromomycin and
tobramycin, respectively, was measured (Fig. 3, A and
B). In the presence of paromomycin, aminoacylation of
tmRNA-TLD slightly increases up to 20 mM
[Mg2+], whereas in the presence of tobramycin,
aminoacylation of tmRNA show almost no difference up to 10 mM [Mg2+]. At higher magnesium
concentrations, the aminoacylation plateau decreases for both RNAs with
and without aminoglycosides, probably because elevated magnesium
concentrations start degrading the RNAs (not shown). Therefore, for
both RNA-aminoglycoside interactions, increasing the magnesium
concentration during the aminoacylation reaction cannot rescue the
inhibitory effect caused by the aminoglycosides on aminoacylation.
Using lead acetate as a probe, we performed a footprinting experiment
between 500 µM neomycin B and labeled tmRNA-TLD at
various magnesium concentrations (1, 2, 5, and 10 mM; not
shown). The result is that the footprints do not depend on magnesium
concentration and therefore strengthen our claim that magnesium does
not rescue aminoglycoside inhibition.
Inhibition of tmRNA Aminoacylation by Neomycin B in the
Presence of Specific Ligands--
SmpB (small
protein B) and EF-Tu·GTP both enhance alanine
accepting activity of tmRNA, and SmpB protects the 3' end of
Ala-tmRNA against enzymatic degradation (28-30). 1 µM of tmRNA with post-transcriptional modifications
charges alanine to 26% on average, but there is a half-increase in
charging when either 1 µM of purified SmpB or 1 µM of purified EF-Tu·GTP is present (Fig.
4, inset). A 2-fold increase
is observed when both proteins are present at 1 µM. This indicates that in our aminoacylation assays, both SmpB and EF-Tu·GTP bind tmRNA and enhance its aminoacylation with alanine. With
EF-Tu·GDP, the charging level of tmRNA is not significantly
affected, but when in the presence of SmpB, the aminoacylation plateau
also increases 2-fold (Fig. 4, inset). We verified by native
gel shift assays that SmpB, EF-Tu·GTP, and EF-Tu·GDP bind
deacylated tmRNA (not shown). In the presence of an equimolar ratio
of SmpB, neomycin B can still inhibit aminoacylation, but the
Ki increases 1.5-fold, compared with tmRNA
alone. In the presence of EF-Tu·GTP, the Ki
increases 5-fold; in the presence of both proteins, the
Ki increases 10-fold, compared with tmRNA alone
(Fig. 4). In the presence of 1 µM EF-Tu·GDP, the
Ki increases 2.5-fold; in the presence of 1 µM SmpB and 1 µM EF-Tu·GDP, the Ki also increases 10-fold, compared with tmRNA
alone (Fig. 4, inset). These data indicate that in the
presence of SmpB and EF-Tu, tmRNA aminoacylation is significantly
protected against the inhibitory effect of aminoglycosides.
Thermal UV Melting Profiles tmRNA-TLD in the Presence of
Aminoglycosides--
UV absorbance melting curves were performed to
assay the effects of aminoglycosides on tmRNA and tmRNA-TLD
structures. In the presence of 5 mM MgCl2, the
melting profile of tmRNA is multiphase, with a first transition
around 40 °C likely corresponding to the unfolding of its tertiary
structure; a series of smaller transitions from 60 to 90 °C
correspond to the progressive unfolding of the secondary structure (not
shown). Because the unfolding pathway of tmRNA is so intricate, no
clear picture emerges, e.g. stabilization or
destabilization, when aminoglycosides are present (not shown). For
tmRNA-TLD, however, UV melting experiments have provided
information about the effects of aminoglycosides on the RNA structure
(Fig. 5). In the absence of
aminoglycosides, tmRNA-TLD structure unfolds in two transitions;
the first one is centered on a calculated melting temperature
(Tm) of around 35 °C; the second transition occurs at higher temperature, with a Tm close to
80 °C (Fig. 5A). That second transition is broad and
noncooperative, suggesting that several conformations of the RNA
coexist in solution and unfold independently when the temperature
increases. 10 mM tobramycin does not affect the lower
transition, whereas the Tm corresponding to the
second transition is considerably reduced (a decrease of 18 °C), to
60 °C (Fig. 5B). Also, the second transition becomes sharper and cooperative. A similar destabilizing effect on
tmRNA-TLD structure is observed in the presence of 2 mM
paromomycin, with no significant effects on the lower transition, but a
16 °C drop of the Tm corresponding to the
second transition (Fig. 5C). As for tobramycin, the second
transition, although shifted to a lower temperature, is nevertheless
sharper and more cooperative compared with tmRNA-TLD alone.
Therefore, both aminoglycosides reduce the conformational freedom of
tmRNA-TLD and stabilize a conformation that is no longer
aminoacylatable with alanine. In the presence of 500 µM
neomycin B, the Tm corresponding to the first
transition is not affected, but the Tm of the
second increases of about 10 degrees, up to 90 °C (not shown).
Therefore, upon binding tmRNA-TLD, neomycin B stabilizes its
conformation.
Chemical Footprints of tmRNA-TLD with Neomycin B--
To gain
structural information about the interaction between tmRNA-TLD and
neomycin B, lead acetate footprints, in the absence and presence of
increasing concentration of neomycin B, were performed (Fig. 3,
C and D). Nine nucleotides
(A8-U12 and
A15-C18) from the D-analog
of tmRNA-TLD and nucleotides G31 and A32
are cleaved by lead but become protected in the presence of increasing concentrations of neomycin B (Fig. 3, C and D).
Six nucleotides involved in the three base pairs
U6-A52, G7-C51, and
G34-C50 are cleaved by lead in the absence of
the aminoglycoside, suggesting that these pairs are unstable in
tmRNA-TLD; when neomycin B is present, all of the six cleavage
sites disappear or are significantly reduced, suggesting that neomycin
B, upon binding tmRNA-TLD, stabilizes the three pairings (result
consistent with the increase of the Tm
corresponding to the second transition). There are no visible changes
in the conformation of stem-loop H5, the five upper pairs in H1, and
the four upper pairs in the T-stem and the T-loop, with and without the aminoglycoside.
Chemical Footprints of tmRNA with Paromomycin and
Tobramycin--
The phosphates (ENU) or nucleotides (lead) of
tmRNA that are protected or that become accessible in the presence
of the aminoglycosides are indexed on the right-hand sides of the
four upper panels (A-D) in Figs.
6 and 7. The
footprints of both aminoglycosides are
summarized on the secondary structure of E. coli tmRNA.
Mapping of ribose accessibility with iron-EDTA shows no differences in
the absence and presence of increasing concentrations of both
aminoglycosides (Figs. 6 and 7, panels E are
representatives; additional experiments were performed with longer
migration times and 3' labeling of the RNA to get resolution of the
upper part of the gels), suggesting that they do not contact any sugars
from tmRNA backbone.
Mapping the phosphates from tmRNA by ENU in the absence of
aminoglycoside shows that of 363 phosphates, 39 are cleaved by the
probe and therefore accessible in the native conformation of tmRNA
(Figs. 6 and 7, A and B). Paromomycin induces 29 concentration-dependent chemical footprints onto tmRNA
structure, 15 against ENU and 14 against lead (Fig. 6,
A-D). With lead, two positions that are not accessible in
tmRNA structure, C44 in helix H2 and G150
in the helical portion of pseudoknot PK2, become reactive in the
presence of paromomycin. Because of the conformational flexibility of
tmRNA (22), several nucleotides located in helical portions within
the RNA structure are cleaved by lead in the absence of the
aminoglycoside. These nucleotides are U26-U27
(5' to an internal bulge within H5), G31-A32
(between two internal bulges in H5, and G31 is involved in
GA pair), U59 (in one stem of PK1), C353 (at
one end of H1), U328-U330 (flexible GU pairs
in H5), and G333 (at one end of H5). When paromomycin binds
tmRNA, these accessible sites become protected (direct contact
between the aminoglycoside and tmRNA or indirect effect of
paromomycin on the conformation of tmRNA). Increasing
concentrations of paromomycin modify the reactivity of nucleotides or
phosphates located in the tRNA-like domain, H5-PK4, PK1-H3-H4, and PK2
but not in H6 and PK3. Within the tRNA-like domain of tmRNA, seven
positions (U9, U17,
U328-U330, G333, and
C353) become protected by paromomycin, as tobramycin also
does but at much higher concentrations (Fig. 7).
Tobramycin induces 31 concentration-dependent chemical
footprints onto tmRNA structure, 14 against ENU and 17 against lead (Fig. 7, A-D). As for paromomycin, C44 in helix
H2 became reactive toward lead cleavage. Three nucleotides, G61 in PK1, G150 in PK2, and G288
in PK4 are protected against both lead and ENU cleavages (Fig. 7,
black stars). The footprints are in the tRNA-like domain,
PK1-H3-H4, and PK2-PK3-PK4.
Paromomycin and tobramycin share 20 common footprints located for the
most part in the tRNA-like domain, the tag reading frame, and PK2. Of
the common footprints between tmRNA and both aminoglycosides, seven
are in the tRNA-like domain, indicating that tight binding requires
full-length tmRNA. This result is in agreement with a higher
Ki for tmRNA-TLD aminoacylation by both
aminoglycosides, compared with tmRNA (Fig. 2 and Table I). However,
there are significant differences between both aminoglycosides
(underlined nucleotides in Figs. 6 and 7): paromomycin, but
not tobramycin, modifies specifically the reactivity of a cluster of
nine nucleotides centered on an internal bulge in H5 and in PK4.
Tobramycin, but not paromomycin, modifies specifically the reactivity
of 11 nucleotides, in PK1, in and around the tag, in PK3 and PK4.
Altogether, our probing data suggest that when aminoglycosides bind
tmRNA, there is a significant conformational rearrangement of the
RNA, as already suggested by our UV melting data collected on
tmRNA-TLD.
Functional and structural evidence is provided to demonstrate that
aminoglycoside antibiotics interact in vitro with tmRNA from E. coli and modify its conformation in solution,
resulting in its inability to be efficiently aminoacylated with alanine by alanyl-tRNA synthetase. tmRNA aminoacylation was most strongly inhibited by neomycin B and by paromomycin followed by, in descending order, gentamicin, amikacin, kanamycin A, and tobramycin (Fig. 2). When
tmRNA is reduced to its tRNA-like domain, the concentration of
paromomycin, gentamicin, and amikacin required for half-inhibition of
aminoacylation increases ~2-fold (Table I), indicating that structural domains of tmRNA outside the tRNA-like core are required for optimal inhibition of aminoacylation. This functional result is
confirmed by our structural analysis of tmRNA in complex with two
different aminoglycosides, because paromomycin and tobramycin both
induce chemical protections of accessible bases and phosphates located
outside the tRNA-like domain (Figs. 6 and 7). Therefore, with the
exception of neomycin B, efficient binding of aminoglycosides requires
full-length tmRNA. Neomycin B interacts specifically with yeast
tRNAPhe (17), rationalizing that the tRNA portion of
tmRNA is sufficient for efficient binding.
Between tmRNA-TLD and tRNAAlaUGC, the concentrations of
paromomycin, gentamicin, and amikacin required for half-inhibition of
aminoacylation also increase ~2-fold (neomycin B is an extreme case,
with a 30-fold difference). Specific sequences and/or structural
features of tmRNA-TLD that are not present in tRNAAla
are responsible. These differences are located in the
D-analog and the C21-G30 stem-loop
of tmRNA-TLD; there are also a few sequence variations in the
acceptor stem and the T stem-loop of canonical tRNAAla,
compared with tmRNA-TLD. Neomycin B protects accessible nucleotides in the D-analog of tmRNA-TLD against lead cleavages
that are not present in tRNAAla, rationalizing the binding
specificity of the aminoglycoside on tmRNA-TLD. For an
aminoglycoside to inhibit tmRNA aminoacylation, it could prevent
the correct recognition of the limited set of nucleotides that specify
the alanine identity, all located in the acceptor stem, by the AlaRS;
alternatively, they could stabilize a nonfunctional state of tmRNA.
Cocrystallization of yeast tRNAPhe with neomycin B reveals
that the aminoglycoside is positioned in the deep groove below the
D-loop, possibly interfering with the interaction between
PheRS and its cognate tRNA through its binding to major
tRNAPhe charging determinants (17). The structural data
reported here indicate that neomycin B interacts with the D-analog of
the tRNA-like portion of tmRNA but not in the upper portion of the
acceptor stem where the major recognition determinant for alanylation, a GU pair, is located. Upon binding, neomycin B strengthens three base
pairs at the junction between the acceptor and the T stems, perturbing
the conformation of the acceptor stem that contains the aminoacylation
signals. When tmRNA-TLD aminoacylation is performed at 20 °C
instead of 37 °C, the Ki for neomycin B increases about 4-fold (from 35 to 145 µM). Neomycin B is a better
inhibitor when the temperature increases, perhaps allowing the RNA to
adopt an optimal conformation for binding.
The footprints of tobramycin and paromomycin on tmRNA and the fact
that aminoglycoside binding can be described in two slopes (at least in
some cases; Fig. 2) suggest that several antibiotic moieties bind to
each tmRNA molecule. Both paromomycin and tobramycin induce the
formation of a favored conformation of tmRNA-TLD, but 7-fold lower
concentrations of paromomycin are required, compared with
tobramycin (Table I). Without aminoglycosides, the
conformation of tmRNA-TLD is poorly defined in solution (Fig. 5),
but aminoglycosides trap tmRNA-TLD into an inactive conformation
that is either stabilized (neomycin B) or destabilized (paromomycin and
tobramycin). Aminoglycosides displace a conformational equilibrium
toward nonfunctional tmRNAs; once the conformational switch is
triggered, increasing the magnesium concentration cannot restore the
aminoacylation capacities of tmRNA (Fig. 3).
tmRNA aminoacylation is required for trans-translation.
It is not known whether or not aminoglycosides bind tmRNA in
vivo. trans-Translation will be impaired in
vivo only if the intracellular concentration of the
aminoglycoside, in our case neomycin B, is sufficient to interact with
some of the ~500 copies of tmRNA/cell (31), in addition to its
other RNA targets. In bacteria, aminoglycosides impair various cellular
processes; they mostly disturb ribosome decoding and cause misreading
of the genetic code (32). Paromomycin binds RNA constructs containing
the ribosomal A site with dissociation constants of ~1.5
µM (33), indicating that the major target of
aminoglycosides is the ribosome. They also inhibit RNase P RNA cleavage
in vitro. The strongest inhibitor of E. coli
RNase P RNA cleavage in the presence of its associated protein is also neomycin B (16), with a Ki value of 60 µM (paromomycin has a Ki value of 190 µM on RNase P RNA). Therefore, the Ki
values of inhibition of RNase P RNA cleavage and tmRNA alanylation
by both neomycin B and paromomycin are similar.
tmRNA aminoacylation with alanine can be impaired by an
aminoglycoside in the presence of all tRNAs from E. coli
(Table III). In its natural context, tmRNA has specific ligands.
Two proteins that interact with tmRNA, SmpB (34) and EF-Tu (35),
stimulate tmRNA aminoacylation in vitro (28). When both
proteins are added to deacylated tmRNA prior neomycin B, they
prevent tmRNA charging from being inhibited by low concentrations
of aminoglycoside (Fig. 4). Whereas SmpB has a small protecting effect,
EF-Tu, in both the GDP and the GTP forms, has a significant protecting
effect against the inhibition of tmRNA aminoacylation by neomycin
B. Protection is maximal when both SmpB and EF-Tu are present,
suggesting that tmRNA aminoacylation might not be impaired in
vivo. RNase T1 footprints of SmpB on a tmRNA
transcript indicate that nucleotides G333 (G31
in tmRNA-TLD) and G336 (G34 in
tmRNA-TLD) are protected by SmpB against RNase T1
cleavage (28). In the presence of neomycin B, G31 from
tmRNA-TLD is protected against lead cleavage and the
G34-C50 pairing is reinforced, indicating that
there are some overlaps between the binding sites of SmpB and neomycin
B on tmRNA. Deacylated tmRNA forms a complex with either
EF-Tu·GDP or EF-Tu·GTP, and two UV cross-links with EF-Tu·GDP are
located outside the tRNA part, in H5 (U268) and PK4
(U308) (36). Binding sites between EF-Tu and neomycin B
onto tmRNA might overlap. Alternatively, SmpB and EF-Tu could
modify the conformation of deacylated tmRNA, with high efficiency
when both proteins are present, such as tmRNA becomes an efficient
substrate for aminoacylation but an inefficient ligand for neomycin B. In the presence of an equimolar ratio of purified ribosomal protein S1
that also interacts with tmRNA, the inhibition constant of tmRNA aminoacylation with neomycin B increases 3-fold (not shown).
All of the known 298 tmRNA gene sequences (20) possess the
recognition determinants for efficient aminoacylation with alanine, suggesting that in all these species the first amino acid of the tag is
alanine. The secondary and probably also tertiary structures of these
tmRNA sequences might be sufficiently conserved to provide the
proper recognition determinants for aminoglycoside binding. Therefore,
aminoglycosides are probably also able to interfere with
trans-translation in bacterial species other than E. coli. Mutating the acceptor stem of tmRNA to confer histidine
acceptance retains its ability of protein tagging in vitro,
suggesting that the first alanine of the tag can be substituted by
another amino acid (37). Therefore, after specific mutations within its
nucleotide sequence, tmRNA could potentially be chargeable by amino
acids other than alanine. Paromomycin and tobramycin modify the
reactivity of the tag reading frame toward structural probes, likely
disturbing reregistration from the stalled ribosome to the tag and
preventing tagging of the truncated proteins, even if aminoacylation
can proceed. The overall pharmacological effect of aminoglycosides during the treatment of infectious diseases may result from a combination of actions prior and during protein synthesis and also when
protein synthesis has stalled or has been interrupted. Aminoglycosides
primarily cause misreading of mRNA that leads to the synthesis of
nonsense or truncated peptides. If tmRNA function is also impaired
by aminoglycosides, the nonsense or truncated peptides will accumulate,
speeding up cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 mCi/mmol) and
[
-32P]pCp (3000 mCi/mmol) were from PerkinElmer Life
Sciences. E. coli tmRNA (22) and
tmRNA-TLD2 were overexpressed in E. coli cells and purified as described. Appropriate bands were
electroeluted, and pure RNAs were recovered by ethanol precipitation.
Total tRNAs from E. coli are from Sigma-Aldrich. Purified
E. coli tRNAAlaUGC was a gift from Dr. R. Gillet
(Medical Research Council, Cambridge, UK).
-mercaptoethanol, 10 mM MgCl2, 2 mM ATP ([MgCl2]/[ATP] = 5), 0.05 mg/ml of
bovine serum albumin, 42-59 µM
L-[14C]alanine (170 mCi/mmol), and 50-330
nM purified E. coli AlaRS. When varying the pH
from 5.5 to 9.5, 50 mM MES was used for pH 5.5, and
50 mM Tris-HCl was used for pH levels from 6.5 to 9.5. Usually, 1 µM of tmRNA or tmRNA-TLD was denatured
for 3 min at 75 °C followed by 10 min at room temperature. Then 1 µM of purified E. coli His6-tagged
SmpB and/or His6-tagged EF-Tu·GDP and/or EF-Tu·GTP were
added and incubated for 15 min at room temperature. All of the proteins
were at least 98% pure as judged by SDS-PAGE analysis. EF-Tu·GDP was
activated to EF-Tu·GTP immediately before use by incubation at
37 °C for 15 min in 50 mM Tris-HCl (pH 7.6), 7 mM MgCl2, 60 mM NH4Cl
complemented with 100 µM GTP, 6 mM
phosphoenolpyruvate, and 10 µg/ml of pyruvate kinase. After the
incubation, the mixture was kept on ice before use. Various
concentrations of aminoglycosides were added, followed by the
aminoacylation buffer, the labeled alanine, and the AlaRS. The aliquots
were spotted onto 3MM Whatman papers at different times, and
precipitated with 5% trichloroacetic acid. Kinetic parameters
(Km and Vmax) in the presence and absence of aminoglycosides were performed under steady-state conditions of enzyme (50-330 nM AlaRS) and substrate
concentrations of tmRNA (0.5-3.4 µM) or
tmRNA-TLD (0.5-6 µM) and determined from Lineweaver-Burk plots. These experimental conditions were also applied
for tmRNA aminoacylation in the presence of all tRNAs from E. coli.
-32P]ATP and
phage T4 polynucleotide kinase on RNA dephosphorylated previously with
alkaline phosphatase. Labeling at their 3' ends was carried out by
ligation of [
-32P]pCp using T4 RNA ligase. After
labeling, tmRNA was gel-purified (5% PAGE), eluted, and
ethanol-precipitated. Labeled tmRNA was heated 2 min at 80 °C
and slowly cooled down for 20 min at room temperature. The reaction
mixtures (15 µl) contained 200,000 cpm of 32P-labeled
tmRNA or tmRNA-TLD and increasing amounts of
aminoglycosides (50-500 µM of neomycin B, 0-1000
µM of paromomycin, and 0-6 mM of tobramycin)
in 50 mM Hepes (pH 7.5), 25 mM KCl, and 2 mM MgCl2. After an incubation of 10 min at room
temperature, ENU, lead-acetate, or iron-EDTA were added: 6.25 µl of a solution of ENU saturated in 100% EtOH supplemented with 1 µg of total tRNA; 0.3 mM of lead acetate supplemented
with 2.5 µg of total tRNA and 1 mg/ml of Fe(NH4)2(SO4)2, 5 mM EDTA, and 12.5 mM dithiothreitol; and 0.25% H2O2 supplemented with 2 µg of total tRNA for
iron-EDTA mapping (a quick centrifuge spin mixed all four chemicals
deposited at four locations inside the Eppendorf tube). The incubation
times were 4 h at 37 °C for ENU, 7 min at 37 °C for lead
acetate, and 10 min at 0 °C for iron-EDTA footprints. RNAs are
ethanol-precipitated, and the pellets are washed twice with 80% EtOH,
dried, and counted. Then the RNA fragments are submitted to 8 or 12%
PAGE. The results were analyzed on a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). During lead mapping of tmRNA in the
presence of either tobramycin or paromomycin, we noticed that gradually
increasing the concentration of the aminoglycosides increases the ratio
of the uncleaved versus the cleaved RNA. Modifying the lead
concentration, the incubation times, or the amount of total RNA added
during the reaction could solve the issue. Because the overall
conformation of tmRNA changes in the presence of both
aminoglycosides, the conformation of tmRNA in the presence of the
aminoglycosides might be less sensitive to the action of the structural probe.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
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Fig. 1.
E. coli tmRNA, tmRNA-TLD
(the tRNA-like portion of tmRNA), and the structures of the
aminoglycosides tested in this study. A, sequence and
secondary structure of E. coli tmRNA (38), with emphasis
on the tRNA-like domain; the additional domains containing four
pseudoknots (PK1-PK4) and the tag reading frame are presented in
outline (B) E. coli tmRNA-TLD; both RNAs have
an alanine attached to their 3' ends. Nucleotides specifying the
alanine identity to both RNAs are circled; in
gray is shown the GU pair, a major identity element for
alanylation. C, structures of the six aminoglycoside
antibiotics used in this study.
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Fig. 2.
Inhibition of tmRNA (gray
circles) and tmRNA-TLD (black squares)
aminoacylation with alanine by six aminoglycoside antibiotics.
Aminoacylation plateaus as a function of increasing concentrations of
neomycin B, paromomycin, gentamicin, amikacin, kanamycin A, and
tobramycin. For a direct comparison between the two RNAs, the
percentage of charging by AlaRS of tmRNA and tmRNA-TLD without
aminoglycosides was set to 100%, whereas tmRNA is aminoacylated up
to 40% and tmRNA-TLD up to 90%. At low concentrations (100-200
µM) of kanamycin A, a beneficial effect on tmRNA
aminoacylation was reproducibly observed.
Ki values of the inhibition of aminoacylation of tmRNA,
tmRNA-TLD, and tRNAAlaUGC by six aminoglycosides
Kinetic parameters of tmRNA-TLD and tmRNA aminoacylation with
alanine in the presence of aminoglycosides
Inhibition of tmRNA aminoacylation with alanine by an
aminoglycoside in the presence of all E. coli tRNAs
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Fig. 3.
A and B, influence of the
concentration of magnesium on the aminoacylation of tmRNA and
tmRNA-TLD, in the presence (gray squares) and absence
(black diamonds) of aminoglycosides. MgCl2:ATP
is at a constant ratio (5:1), whereas the magnesium concentration
increases. A, aminoacylation of tmRNA-TLD in the
presence of 500 µM paromomycin (10% of variance without
and with the aminoglycoside). B, aminoacylation of tmRNA
in the presence of 2 mM tobramycin (10% of variance
without and with the aminoglycoside). C and D,
lead acetate footprints of tmRNA-TLD with increasing concentrations
of neomycin B. C, autoradiograms of 12% denaturing PAGE of
cleavage products of 3'-labeled RNAs. Lane C, incubation
control; lane GL, RNase T1 hydrolysis
ladder; lane AL, RNase U2 hydrolysis
ladder. The RNA sequence is indexed on the left side.
Gray nucleotides indexed on the right side are
the identified protections. D, nucleotides from
tmRNA-TLD with a decreased reactivity toward lead cleavage in the
presence of neomycin B are in gray. The major recognition
determinant for alanylation, a GU pair, is boxed.
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Fig. 4.
Inhibition of tmRNA aminoacylation with
alanine by neomycin B in the presence of purified SmpB, EF-Tu·GTP, or
both. Aminoacylation plateaus as a function of increasing
concentrations of neomycin B of 1 µM tmRNA
(T, diamonds); 1 µM tmRNA and 1 µM SmpB (T+S, squares); 1 µM tmRNA and 1 µM EF-Tu·GTP
(T+E, triangles); and 1 µM
tmRNA, 1 µM SmpB, and 1 µM EF-Tu·GTP
(T+E+S, circles). For a direct comparison, the
percentage of charging by AlaRS of tmRNA with and without protein
ligands in the absence of aminoglycoside was set to 100%.
Inset, charging levels and inhibition constants of
aminoacylation of tmRNA with and without SmpB and/or EF-Tu in both
the GDP and the GTP forms. The plots and values reported are averaged
from four independent experiments for each condition.
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Fig. 5.
Thermal UV melting profiles tmRNA-TLD in
the presence of various aminoglycosides. A, thermal UV
melting profiles of tmRNA-TLD. The variance in the calculated
Tm was calculated from 13 independent
experiments. B, UV absorbance melting profile of
tmRNA-TLD in the presence of 10 mM tobramycin. The
variance in the calculated Tm was calculated
from six independent experiments. C, UV absorbance melting
profile of tmRNA-TLD in the presence of 2 mM
paromomycin. The variance in the calculated Tm
was calculated from six independent experiments. The first
derivative of the UV absorbency as a function of temperature is also
shown. The plots shown are averaged from three independent
experiments.
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Fig. 6.
Chemical footprints of increasing
concentrations of paromomycin with tmRNA using ENU (A
and B), lead acetate (C and
D), and iron-EDTA (E).
Autoradiograms of 8% denaturing PAGE of cleavage products of 5' and 3'
labeled tmRNAs, with similar migration times. Lanes C,
incubation controls; lanes GL, RNase T1
hydrolysis ladder; lanes AL, RNase U2
hydrolysis ladder. The sequence is indexed on the left
sides. Nucleotides with black and white
triangles are Gs and As, respectively. Nucleotides indexed on the
right sides of the autoradiograms are the identified
footprints. Footprints of the aminoglycoside are indicated onto
tmRNA secondary structure, shown schematically. Only the
protections or enhancements of reactivity of nucleotides that vary
according to the concentration of the aminoglycoside were considered as
reliable data. The black dots are the phosphates protected
against ENU cleavages in the presence of the aminoglycoside. The
gray dots are the nucleotides protected against lead
cleavage in the presence of the aminoglycoside. The black
stars are the positions that are protected by both ENU and lead in
the presence of the aminoglycoside. The gray squares are the
nucleotides that become accessible to lead cleavage in the presence of
the aminoglycoside. With ENU, some nucleotides become accessible in the
presence of the aminoglycoside, but all of these cleavage sites are
already present in the control lane; thus, they were omitted on
purpose. The footprints that are specific to the aminoglycoside are
underlined (those that are common to paromomycin and
tobramycin are not).
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Fig. 7.
Chemical footprints of increasing
concentrations of tobramycin with tmRNA. The indications
provided are identical to those in Fig. 6.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
In the lab, we thank Dr. L. Metzinger for cloning E. coli SmpB and Dr. M. Hallier for advice on protein purifications. We thank Drs. R. Gillet and F. Murphy (Medical Research Council, Cambridge, UK) for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was funded by Human Frontier Science Program Research Grant RG0291/2000-M 100, by a Research Grant entitled "Recherche Fondamentale en Microbiologie et Maladies Infectieuses," and by an Action Concertée Incitative Jeunes Chercheurs 2000 from the French Ministry of Research (to B. F.).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.
The on-line version of this article (available at
http://www.jbc.org) contains supplemental data on the chemical footprints.
Supported by a Projet de Recherche d'Intérêt
Régional grant from the region of Brittany.
§ To whom correspondence should be addressed. E-mail: bfelden@univ-rennes1.fr.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M212830200
2 C. Gaudin, S. Nonin-Lecomte, C. Tisné, S. Corvaisier, V. Bordeau, F. Dardel, and B. Feldon, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: tmRNA, transfer messenger RNA; tRNA, transfer RNA; EF-Tu, elongation factor Tu; AlaRS, alanyl-tRNA synthetase; MES, 4-morpholineethanesulfonic acid; TLD, tRNA-like domain; ENU, ethyl nitroso urea.
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REFERENCES |
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