(Received for publication, August 14, 1995)
From the
A molecular basis for the insensitivity of eukaryotic ribosomes
to the antibiotic thiostrepton was investigated using synthetic
100-nucleotide-long fragments covering the GTPase domain of 23/28 S
rRNA. Filter binding assay showed no detectable binding of the rat RNA
to thiostrepton, but the binding capacity was markedly increased by
base substitution of G to A at the position
corresponding to 1067 of Escherichia coli 23 S rRNA. The
association constant (K
) for the rat
A
mutant was 0.60
10
M
, which was comparable with that of the E. coli RNA (K
= 1.1
10
M
). This suggests
that the eukaryotic G
participates in the resistance for
thiostrepton. On the other hand, the RNA fragments of the two species
had a similar binding capacity for E. coli ribosomal protein
L11 and its mammalian homologue L12. Gel electrophoresis under a high
ionic condition, however, revealed a difference between the two
proteins. E. coli L11 formed stable complexes with both the E. coli RNA and the rat A
mutant RNA in the
presence of thiostrepton, while rat L12 failed to exhibit such complex
formation. This suggests that the eukaryotic L12 protein may also be an
element giving the resistance for thiostrepton. These results are
discussed in terms of preserved three-dimensional conformation of the
RNA backbone between prokaryotes and higher eukaryotes.
A number of antibiotics bind to prokaryotic ribosomes and
interfere with protein synthesis. These antibiotics have been used as
powerful tools for dissecting the translational
mechanism(1, 2, 3) . Thiostrepton, one of
such compounds, binds tightly to the 50 S ribosomal subunit with 1:1
stoichiometry (4) and inhibits ribosome-associated GTPase
events(2) . The primary target of thiostrepton lies within a
limited region comprising residues 1052-1112 in domain II of 23 S
rRNA, termed the ``GTPase domain'' (5, 6) .
Binding of thiostrepton to this RNA domain is greatly enhanced by an
association of Escherichia coli ribosomal protein L11 with a
region encompassing the thiostrepton site(7) , presumably by
stabilizing and/or adjusting RNA structure(8) . The residue
A in this domain plays an important role in the drug
binding, i.e. methylation of the 2`-O of this residue renders
ribosomes resistant to thiostrepton(9) ; base substitutions at
this residue reduce the affinity for
thiostrepton(6, 10, 11, 12) ; the
antibiotic protects the N1 position of A
from chemical
modification(5) . On the other hand, the base A
is known as a plausible site of direct interaction with
elongation factor EF-G(13, 14) .
Eukaryotic
ribosomes are totally resistant to thiostrepton despite a high level of
conservation of primary and secondary structure at the target RNA
region(15) . It is noteworthy that the base at the position
equivalent to E. coli A is replaced to G in the
eukaryotic GTPase domain. This G base is specifically protected from
chemical modification by the binding of eukaryotic elongation factor
EF-2(16) . Furthermore, the G base is the element required for
recognition by anti-28 S autoantibody, which has a preference for the
eukaryotic GTPase domain(16) . These data imply that the G base
at the 1067 equivalent position is an important identity element of the
eukaryotic domain that is involved in the resistance for thiostrepton.
To know whether this is the case, we tested an effect of substitution
of A for the G base in the eukaryotic RNA domain on the thiostrepton
binding. Here, we show that the binding affinity of the rat RNA for
thiostrepton increases to a level comparable to that of E. coli wild type by a single G to A substitution. Also, effects of L11
and its mammalian homologue L12 on the drug binding are presented.
The prokaryotic 50 S ribosomal
subunits were prepared from E. coli W3110 strain, as described
previously(19) . The total proteins (TP50) were extracted from
the subunits with 66% acetic acid, 33 mM MgCl(20) and recovered by precipitation with 7 volumes of
cold acetone. The TP50 was fractionated by a stepwise elution from
CM-cellulose (Whatman) column equilibrated with a buffer (6 M urea, 5 mM 2-mercaptoethanol, and 20 mM sodium
acetate, pH 4.6). A protein fraction enriched with E. coli L11
was eluted with the same buffer containing 75 mM LiCl. L11 was
further purified by high performance ion-exchange chromatography; the
protein fraction was applied to a CM-5PW column (Tosoh) equilibrated
with a buffer consisting of 6 M urea, 5 mM 2-mercaptoethanol, 40 mM LiCl, and 20 mM sodium
phosphate, pH 6.5, and eluted with a linear gradient of 40-200
mM LiCl. Purity and identity of the final L11 sample was
ascertained by two-dimensional polyacrylamide gel
electrophoresis(19) . The proteins were concentrated with
Centricon-10 (Amicon) and dialyzed against the renaturation buffer (300
mM KCl, 5 mM 2-mercaptoethanol, and 20 mM Tris-HCl, pH 7.5).
Synthetic small RNA fragments covering the GTPase domain of E. coli 23 S rRNA bind to thiostrepton with almost the same
affinity as full-length 23 S rRNA(6, 11) . An RNA
fragment containing residues 1841-1939 of rat 28 S rRNA (Fig. 1A) and one containing residues 1029-1127
of E. coli 23 S rRNA (Fig. 1B) were
synthesized using a SP-6 transcription system, and the two RNAs were
subjected to a filter binding assay. Fig. 2A shows a
titration of the E. coli wild-type RNA with thiostrepton. The
apparent association constant was 1.1 10
M
as determined by double reciprocal plot
of the binding data (Fig. 2C). This is comparable to
values previously estimated by other workers using either the
equilibrium dialysis (2) or the filtration
technique(11) . Rat wild-type RNA, however, showed no
detectable binding at concentrations up to 2 µM of
thiostrepton (Fig. 2B) and even at 16 µM (not shown). The binding could not be tested at concentrations
higher than 16 µM because of a low solubility of
thiostrepton. Hence, we failed to determine the binding constant.
Figure 1: Secondary structure models of the GTPase domain from rat 28 S rRNA (A) and E. coli 23 S rRNA (B). The regions of residues 1841-1939 of rat 28 S rRNA (A) and residues 1029-1127 of E. coli 23 S rRNA used in this study are shown. Bases exchanged between the two RNAs are indicated by arrows. The numbering of residues in rat 28 S rRNA is as described(18, 34) .
Figure 2:
Effect of base substitutions on the
thiostrepton binding: A to G of the E. coli RNA (A) and G
to A of the rat RNA. A and B, thiostrepton binding curves for the E. coli RNA
fragment and its G
variant (A) and the rat RNA
fragment and its A
variant (B). Increasing
concentrations of thiostrepton were incubated with 0.1 µM of each
P-labeled RNA: the E. coli wild-type
RNA fragment (E. coli-WT,
), the E. coli G
variant (E. coli-G,
), the rat
wild-type RNA fragment (Rat-WT,
), and the rat
A
variant (Rat-A,
). Each reaction
mixture was applied to a nitrocellulose filter. Binding is expressed as
retention, corresponding to a ratio between the radioactivity retained
on the filter and the input(25) . C, double reciprocal
plot of RNA-thiostrepton binding data. The reciprocal of retention
given in A and B (the mean of three experiments) was
plotted as a function of the reciprocal of the thiostrepton
concentration. The apparent association constants (K
) were estimated from slopes of lines
for the E. coli wild-type RNA (E. coli-WT,
) as
1.1
10
M
and for the
rat A
RNA (rat-A,
) as 0.6
10
M
, based on a relation of
slope =
1/K
(35) .
Data from footprinting (5) and site-directed mutagenesis (11) suggest that bases A, G
,
G
, A
, and A
within the E. coli domain interact with thiostrepton directly. Among
these bases only A
is unique to prokaryotic RNA domain;
the equivalent position in eukaryotic 28 S rRNA has G (G
in rat). The other bases are preserved between prokaryotes and
eukaryotes (see Fig. 1). We therefore reasoned that the
eukaryotic G
at the equivalent position of E. coli 1067 site may be a key element giving the resistance for the
thiostrepton binding. To test this, we synthesized RNAs with base
substitutions of A
to G in E. coli RNA and
G
to A in rat RNA and examined their effects on
thiostrepton binding. The A
to G substitution in E.
coli RNA significantly weakened the binding affinity; no
detectable binding was observed in the filter binding analysis used
here (Fig. 2A). The reciprocal substitution, G
to A in rat RNA, greatly increased the binding capacity (Fig. 2B). The estimated K
value
for the rat A
variant was 0.6
10
M
(Fig. 2C), which was
close to the value for the E. coli wild type (1.1
10
M
). The results indicate
that this substitution results in the up-mutation of thiostrepton
binding and suggest that the A
base participates in the
drug binding.
The RNA fragments were also tested for their binding
capacity to E. coli ribosomal protein L11 (Fig. 3A) and its rat homologue L12 (Fig. 3B) by filter binding assay. E. coli L11
bound not only to the E. coli RNA (K = 1.0
10
M
) but also to the rat RNA (K
= 0.44
10
M
). Similarly, rat L12 bound to both the
rat RNA (K
= 1.4
10
M
) and the E. coli RNA (K
= 1.5
10
M
) with almost the same affinity. Either of
the base substitutions, E. coli A
to G and rat
G
to A, gave no significant effect on the binding of E. coli L11 and rat L12 (not shown). In these protein binding
experiments, we used rat S1 RNA variant (18) as a negative
control. This RNA contains a mutation of U
to A, which
leads to disruption of rat L12 binding(18) , probably due to
perturbation of the conserved bulge structure.
Figure 3:
Binding of ribosomal proteins to the RNA
fragments. Increasing concentrations of E. coli L11 protein (A) and rat L12 protein (B) were incubated with the E. coli wild-type RNA (), the rat wild-type RNA
(
), and rat S-1 variant (
). The S-1 variant has A in the position of U
and used as a negative binding
probe (see text). RNA-protein binding was analyzed as described in the
legend for Fig. 2.
It has been shown
that the binding of E. coli L11 protein to the GTPase domain
greatly enhances the accessibility to thiostrepton and that
thiostrepton is also able to stabilize the binding of L11(7) .
To analyze such cooperativity of the RNAL11
thiostrepton
complex, we used a gel mobility shift analysis in an electrophoresis
buffer containing 0.3 M KCl. Despite this high ionic
condition, a clear band of the complex was observed in a gel by mixing
the three components (Fig. 4, lane 4). In the absence
of the antibiotic, however, the RNA
L11 complex itself was not
detected as a discrete band, presumably due to dissociation of the
complex during electrophoresis (Fig. 4, lane 3). Lane 6 shows that rat L12 protein was not able to substitute
for E. coli L11 in the formation of such a stable complex,
indicating that the rat protein may not cooperate with the RNA and
thiostrepton despite its high affinity for the E. coli RNA (Fig. 3B).
Figure 4:
Gel retardation analysis of the
RNA-protein complex stabilized with thiostrepton. The E. coli wild-type P-labeled RNA was incubated at various
combinations of the ribosomal proteins and thiostrepton, as indicated above gel lanes: without protein (lanes 1 and 2), with 0.25 µg of E. coli L11 (lanes 3 and 4), or 0.3 µg of rat L12 (lanes 5 and 6). The incubation was done in the presence of 20 pmol of
thiostrepton (lanes 2, 4, and 6) or in the
absence (lanes 1, 3, and 5). The complexes
were analyzed on 6% nondenaturing polyacrylamide gels containing 0.3 M KCl, 10 mM MgCl
, and 50 mM Tris-HCl, pH 8.0.
The rat RNA was also tested for forming
the complex with E. coli L11 and thiostrepton. As shown in Fig. 5, rat wild-type RNA formed no stable complex in the gel (Fig. 5, lane 4). However, the substitution of A for
G gave a discrete complex surviving gel electrophoresis (Fig. 5, lane 8). These results show that the G to A
substitution at the 1067 equivalent position makes the eukaryotic RNA
highly accessible to thiostrepton and E. coli L11 in a
cooperative manner.
Figure 5:
Effect of the rat base G to
A substitution on stabilization of the RNA
L11 complex with
thiostrepton. The rat wild-type RNA (lanes 1-4) and its
A
variant (lanes 5-8) were incubated at
various combinations of thiostrepton and E. coli L11, as
indicated above gel lanes: without protein (lanes 1, 2, 5, and 6), with E. coli L11 (lanes 3, 4, 7, and 8) in the
presence of 20 pmol of thiostrepton (lanes 2, 4, 6, and 8), or its absence (lanes 1, 3, 5, and 7). The complex was analyzed on
gels, as described in the legend of Fig. 4.
The secondary structure of the GTPase domain has been deduced by comparing 23 S-type RNA sequences from diverse organisms(5, 11, 15) . Fig. 1shows models for the domain of rat 28 S rRNA (A) and E. coli 23 S rRNA (B), both of which represent a conserved four-loop/four-helix structure. The minimal binding site for thiostrepton comprises residues 1052-1112 sequence of E. coli 23 S rRNA (6) to which ribosomal protein L11 also binds(26) . Among these 61 residues, there are 26 bases different between the E. coli and the rat domain (Fig. 1). Some of the different bases are assumed to be responsible for the low affinity of the rat RNA to the drug. The present data demonstrate that an exchange of the eukaryote-specific base G to A at the single position equivalent to 1067 of E. coli 23 S rRNA increases the affinity for thiostrepton to a level comparable to that of E. coli wild-type RNA.
The
significance of A residue in the thiostrepton binding
has been shown by several approaches using E. coli ribosomes.
A footprinting study by Egebjerg et al.(5) showed
that 10 bases including A
are protected from chemical
attacks. An extensive mutational analysis by Ryan et al.(11) showed that among these 10 protected bases,
substitutions at the following 5 positions gave considerable reduction
of the drug binding: A
, G
,
G
, A
, and A
. These results
lead to an implication that the 5 bases are major sites of direct
contacts with thiostrepton. Among these 5 bases, only A
base is different from G of the eukaryotic domain; the others are
preserved between E. coli and rat at respective positions,
implying that A
is an important element for the
specificity of thiostrepton-RNA binding. Reduction of thiostrepton
binding by base substitution at A
(6, 10, 11, 12) obviously supports
this view. In contrast to disruption analyses of this kind exhibiting
``down-mutation,'' we have used here the reverse approach
making the eukaryotic RNA highly accessible to thiostrepton, i.e. ``up-mutation'' analysis. By the G to A substitution at
the 1067 equivalent position of rat RNA, the thiostrepton binding is
markedly enhanced (Fig. 2B). This result is more
straightforward to explain a key role of A
in
thiostrepton binding.
Cundliffe provided a hypothesis that the
GTPase RNA domain has multiple functional conformations and that
thiostrepton may lock the RNA in a single conformation(2) .
From the data by footprinting that the bases protected by thiostrepton
are concentrated in two loop regions of residues 1065-1073 and of
residues 1093-1098, Egebjerg et al.(27) proposed a model of the three-dimensional
conformation of the GTPase domain, in which the two loops are folded
close together to construct the drug binding site. By this binding, a
single conformation may be rigidly locked, as suggested by
Cundliffe(2) . In a previous study(16) , we showed this
three-dimensional model is also available for the eukaryotic RNA domain
using another ligand, anti-28 S autoantibody specific for the
eukaryotic GTPase domain(24) . Anti-28 S antibody protects 4
bases within the two loop regions of the eukaryotic domain
corresponding to positions 1066, 1067, 1068, and 1098 of E. coli 23 S rRNA(16) . Unlike thiostrepton, anti-28 S antibody
shows an affinity for the prokaryotic GTPase domain much lower than
that for eukaryotic one. However, the binding affinity for E. coli RNA is greatly enhanced by substitution of G for
A(16) . From such interchangeablity of the
ligand specificity by a single base exchange, it is highly probable
that these bases are important identity elements discriminated by
anti-28 S and thiostrepton and that the basal structure of the ligand
binding site is fairly preserved between prokaryotes and higher
eukaryotes as far as protein-free RNA state is concerned.
E.
coli ribosomal protein L11 has been suggested to recognize the
backbone of the GTPase domain in E. coli 23 S rRNA (11, 28) and stabilize the tertiary structure. The
protein also binds to the RNA from archaebacteria (29) and
yeast and mouse(30) . We used this E. coli protein and
its mammalian homologue L12 (31) as probe for study of the
domain structure. The observed close K values of
both proteins for E. coli and rat RNAs (Fig. 3)
indicate the extreme conservation of backbone structure between
prokaryotes and higher eukaryotes. This may explain previous results
that the GTPase domain of E. coli and the yeast equivalent
region can be exchanged with each other without drastic effect of
ribosome function(32, 33) .
The binding affinity of
thiostrepton for the intact E. coli 50 S subunit is extremely
higher than that for 23 S rRNA. This is attributed to the cooperativity
between thiostrepton and E. coli protein L11(7) . It
is suggested that L11 conducts a fine tuning of the RNA tertiary
structure for the cooperativity. This high affinity of thiostrepton for
the intact ribosome is reduced only modestly, when A is
changed to the eukaryotic base G (10) and also when whole
domain containing residues 1056-1103 is exchanged with the yeast
equivalent domain(33) . These data imply that the total
resistance of eukaryotic ribosomes to thiostrepton is not solely due to
eukaryote-specific RNA elements including G base at the A
equivalent position but also to protein components of the
eukaryotic ribosome. Eukaryotic homologues of E. coli L11 are
most likely to be related to the resistance, although no direct
evidence has been represented.
To study the cooperativity between
the L11 homologues and thiostrepton in the RNA binding, we used a gel
electrophoretic analysis under high ionic condition. This gel system
clearly discriminated a rigidly formed RNAL11
thiostrepton
complex from the RNA
L11 complex (Fig. 4). In the gel
analysis, rat L12 failed to form the stable
RNA
L12
thiostrepton complex (Fig. 4) despite its high
affinity for the E. coli GTPase domain (Fig. 3B). This suggests that RNA tuning required for
the cooperativity may not be provided by rat L12. It is noteworthy that
rat A
mutant RNA also forms such a stable complex with
thiostrepton and E. coli L11 but not with rat L12 (data not
shown). As for the eukaryotic domain-specific ligand anti-28 S, a
similar but contrasting result was obtained; rat L12 enhanced the
binding of anti-28 S to the rat GTPase domain. (
)This may be
ascribed to its ability to adjust the structure to eukaryotic type.
Therefore, we think it is possible that the ligand affinity strongly
depends on the kind of associated protein, in addition to the base at
the 1067 position (Fig. 6). The RNA structure itself can be
rather flexible, and its binding to prokaryotic L11 and eukaryotic L12
may make up slightly different RNA conformations that have preferences
for thiostrepton and anti-28 S, respectively.
Figure 6: Schematic representation of the contrasting properties of thiostrepton and anti-28 S autoantibody. The prokaryotic A base at the position 1067 of E. coli 23 S rRNA and eukaryotic G base at the equivalent position are indicated with large letters. An importance of the prokaryotic A base for the binding specificity of thiostrepton (this study) and the eukaryotic G base for that of anti-28 S (our previous study, see (16) ) has been verified by experiments using reciprocal base substitution mutants. Bindings of E. coli L11 and rat L12, which are involved in the specificity of ligand bindings, are represented.