2620–2627 Nucleic Acids Research, 2002, Vol. 30, No. 12
© 2002 Oxford University Press
Interaction among silkworm ribosomal proteins P1, P2
and P0 required for functional protein binding to the
GTPase-associated domain of 28S rRNA
Tomomi Shimizu, Masao Nakagaki1, Yoshinori Nishi2, Yuji Kobayashi2, Akira Hachimori and
Toshio Uchiumi*
Institute of High Polymer Research and 1Department of Applied Biological Science, Faculty of Textile Science and
Technology, Shinshu University, Ueda 386-8567, Japan and 2Graduate School of Pharmaceutical Sciences,
Osaka University, Osaka 565-0871, Japan
Received March 11, 2002; Revised and Accepted April 30, 2002
ABSTRACT
Acidic ribosomal phosphoproteins P0, P1 and P2 were
isolated in soluble form from silkworm ribosomes and
tested for their interactions with each other and with
RNA fragments corresponding to the GTPaseassociated
domain
of
residues
1030–1127
(Escherichia coli numbering) in silkworm 28S rRNA
in vitro. Mixing of P1 and P2 formed the P1–P2
heterodimer, as demonstrated by gel mobility shift and
chemical crosslinking. This heterodimer, but neither P1
or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1–P2)2, assumed
from its molecular weight derived from sedimentation
analysis. Complex formation strongly stimulated
binding of P0 to the GTPase-associated RNA domain.
The protein complex and eL12 (E.coli L11-type), which
cross-bound to the E.coli equivalent RNA domain, were
tested for their function by replacing with the E.coli
counterparts L10.L7/L12 complex and L11 on the rRNA
domain within the 50S subunits. Both P1 and P2,
together with P0 and eL12, were required to activate
ribosomes in polyphenylalanine synthesis dependent
on eucaryotic elongation factors as well as eEF-2dependent GTPase activity. The results suggest that
formation of the P1–P2 heterodimer is required for
subsequent formation of the P0(P1–P2)2 complex and
its functional rRNA binding in silkworm ribosomes.
INTRODUCTION
It is generally accepted that ribosomal proteins modulate the
structure and function of rRNAs (1). The acidic stalk protein
complex, L10.L7/L12 in procaryotes, binds to a region around
residues 1030–1127 in domain II of 23S rRNA, termed the
GTPase-associated domain (2–4), and constitutes a part of the
functional center, termed the GTPase center (5) or factorbinding center (6). Acidic L7/L12 protein (L7 differs from L12
by an acetylated N-terminus) is an important component of this
functional center (7), although there is no evidence for its direct
binding to rRNA. This protein has characteristic properties: there
are four copies, two homodimers, of L7/L12 per ribosome (8);
they are flexible in the ribosome (9–11). The four copies of
L7/L12 bind to L10 and form the pentameric complex (12); the
L10 moiety of the complex appears to bind directly to the
rRNA domain (13). Although it is well known that L7/L12
within the GTPase center participates in interaction of the
ribosome with translation factors (G-proteins) (7,14), the functional significance of the four copies has been poorly understood (15–17).
The structure and function of the acidic phosphoproteins in
eucaryotic ribosomes have been investigated mainly in rat
(18–20), human (21), Artemia salina (22) and yeast (23,24).
Animal ribosomes contain two kinds of the acidic proteins, P1
and P2 (reviewed in 25), whereas yeast ribosomes contain two
P1-type proteins, P1α and P1β, and two P2-type proteins, P2α
and P2β (24). These proteins bind to P0 protein, the equivalent
of Escherichia coli L10, and presumably form the pentameric
complex in the ribosome (22,26), designated here P0.P1/P2.
The P0.P1/P2 complex binds to the GTPase-associated domain
of 28S rRNA (27) and plays a crucial role in kingdom-specific
interaction between 80S ribosomes with eucaryotic elongation
factor 1α (eEF-1α) and elongation factor 2 (eEF-2) (28).
It is interesting that the eucaryotic ribosomes have more than
two kinds of acidic proteins, unlike procaryotic ribosomes.
There are two major interpretations for the states of P1 and P2
in the ribosome: (i) P1 and P2 form homodimers, as suggested
by crosslinking (22,29); or (ii) they form heterodimers,
suggested by yeast two-hybrid system (26,29,30) and the other
genetic and biochemical studies (26,31–33). Isolated P1 and
P2 proteins have a tendency to form oligomeric complexes
(29), which may occur because of non-specific interactions.
The instability of free P1 and P2 proteins in solution seems to
bring ambiguity to the biochemical data. Moreover, the insolubility of isolated P0 protein makes in vitro binding experiments
difficult.
We here used the ribosomal proteins from the silkworm
Bombyx mori; proteins P1, P2 and P0 could be isolated in
soluble form and were used for a study on their interaction
in vitro. We show that formation of a P1–P2 heterodimer is
significant for the subsequent assembly of a pentameric
complex, probably in the form P0(P1–P2) 2, and then its rRNA
*To whom correspondence should be addressed at: 3-15-1, Tokida, Ueda City, Nagano Prefecture 386-8567, Japan. Tel: +81 268 21 5575; Fax: +81 268 21 5571;
Email: [email protected]
Nucleic Acids Research, 2002, Vol. 30, No. 12 2621
binding. To evaluate the in vitro binding data on the basis of
ribosome function, we used a hybrid ribosome system developed
recently (28), in which E.coli L10.L7/L12 complex and L11 on
the 50S subunit were replaced with the eucaryotic counterparts
P0.P1/P2 complex and eL12, respectively. The functions of the
hybrid ribosomes carrying the silkworm ribosomal proteins
were tested with eucaryotic elongation factors. The results
provide evidence that formation of P1–P2 heterodimers is
crucial for their binding to P0 and the subsequent interaction
with the GTPase-associated domain of rRNA, from which
derives factor-dependent ribosome function. We discuss an
additional functional role of the P1–P2 heterodimer as a
modulator of the RNA-binding protein P0.
MATERIALS AND METHODS
Silkworm ribosomes and ribosomal proteins
The high KCl/puromycin-treated 80S ribosomes were isolated
from posterior silk glands of last instar larvae of B.mori (strain
C132), according to the method previously described (34).
Total proteins were extracted from the ribosome in 66% acetic
acid, 33 mM MgCl2 and recovered by precipitation with 7 vol
of cold acetone. The proteins were dialyzed against buffer A
containing 20 mM sodium acetate, pH 4.5, 7.5 M urea and 5 mM
2-mercaptoethanol and then applied to a column of CM-cellulose
(Whatman) equilibrated with the same buffer. Proteins were
successively eluted with buffer A containing increasing
concentrations of LiCl; P1, P2 and P0 were eluted in solutions
containing 0, 0.05 and 0.08 M LiCl, respectively. The protein
fractions were concentrated with Centricon YM-10 (Amicon).
P1 and P2 fractions were dialyzed against buffer B containing
20 mM sodium phosphate, pH 6.5, 6 M urea, 100 mM LiCl, 5
mM 2-mercaptoethanol and further purified by ion exchange
high performance liquid chromatography (HPLC) with
DEAE-5PW (Tosoh) in a linear gradient of 100–300 mM
LiCl. The P0 fraction was dialyzed against buffer C (buffer B
except that the concentration of LiCl was 50 mM) and purified by HPLC with CM-5PW (Tosoh) in a linear gradient of
50–250 mM LiCl. The identities of the P1, P2 and P0 proteins
were confirmed by reactivity with anti-P monoclonal antibody
(35). P1 and P2 were also tested for partial amino acid sequencing
using a protein sequencer (model PPSQ-21; Shimadzu).
Complex formation of P0, P1 and P2
Isolated proteins in the presence of 6 M urea were mixed
together at a molar ratio of P0:P1:P2 of 1:3:3 and dialyzed
against 0.3 M KCl, 20 mM Tris–HCl, pH 7.6, at 0°C. The same
dialysis was also performed in the absence of P1 or P2. The
P1–P2 complex was formed by mixing various ratios of P1 and
P2 in 0.3 M KCl, 20 mM Tris–HCl, pH 7.6, at 0°C. Complex
formation was confirmed by 6% native PAGE (acrylamide/
bisacrylamide ratio 40:1) at 6.5 V/cm with a buffer system
containing 5 mM MgCl2, 50 mM KCl and 50 mM Tris–HCl,
pH 8.0. Samples were electrophoresed for 10 h at constant
voltage and 4°C with buffer recirculation. The gel was stained
with Coomassie Brilliant Blue. Bands of the complexes were cut
out of the gel and the constituents were separated by SDS–PAGE
as described by Laemmli (36), except that the gel contained
22% polyacrylamide and 0.44% bisacrylamide to improve
separation of P1 and P2. The P0, P1 or P2 proteins on the gel
were identified by immunoblotting using an anti-P monoclonal
antibody that cross-reacts with the silkworm proteins. In some
experiments, the P0.P1/P2 complex was further purified by gel
filtration with G-3000SW XL (Tosoh) in a solution consisting of
100 mM KCl, 0.2 mM dithiothreitol, 20 mM Tris–HCl, pH 7.6.
Gel retardation assays
RNA fragments containing residues 1030–1127 of E.coli 23S
rRNA and the equivalent region of B.mori 28S rRNA were
synthesized with SP-6 RNA polymerase using cDNA as
template (34). The transcripts were purified by gel filtration on
a Sephadex G-50 column (Amersham Pharmacia). A solution
(10 µl) containing 5 pmol [32P]RNA fragments, 20 mM MgCl2,
0.3 M KCl and 20 mM Tris–HCl, pH 7.6, was preincubated at
65°C for 5 min and then cooled to 30°C over 10 min. After the
addition of a protein sample as indicated in the legend to
Figure 5, the mixture was further incubated at 30°C for 10 min.
RNA–protein binding was examined by 6% native PAGE
under the same conditions as described above.
Functional assay using the hybrid system
Escherichia coli ribosomes deficient in L11 were obtained
from strain AM68 (37), as described previously (28). The 50S
subunits were incubated in a solution containing 50% ethanol
and 0.5 M NH4Cl to remove specifically the L10.L7/L12
complex, as described in a previous report (28). The 50S core
subunits deficient in L10.L7/L12 and L11 thus obtained were
used to study function of the P0.P1/P2 complex. The 50S core
subunits were incubated with the silkworm ribosomal proteins
P0.P1/P2 (or samples without P1 and/or P2) and eL12, as
described in the legend to Figure 7. The resultant E.coli–silkworm hybrid 50S subunit (0.13 µM) was tested for eucaryotic
eEF-2-dependent GTPase activity in a solution (20 µl)
containing 0.38 µM 30S subunit, 0.25 µM eEF-2, 150 µM
[γ- 32P]GTP, 10 mM MgCl2, 50 mM NH 4Cl, 20 mM Tris–HCl,
pH 7.6, and 0.2 mM dithiothreitol. The reaction was performed
at 37°C for 10 min. Polyphenylalanine synthetic activity was
assayed in a solution (100 µl) containing 0.1 µM hybrid 50S
subunit, 0.5 µM 30S subunit, 10 µg poly(U), 0.4 µM E.coli
[14C]Phe-tRNA (with 80 µg deacylated total tRNA), 200 µM
GTP, 10 mM MgCl2, 75 mM NH 4Cl, 50 mM Tris–HCl, pH 7.6,
0.2 mM dithiothreitol and 800 µg silk gland cytosol fraction as
a source of elongation factors that was obtained by ammonium
sulfate precipitation (40–60%) of the S200 fraction. The reaction was performed at 37°C for 10 min.
Chemical crosslinking
A mixture of P1 and P2 (9.5 nmol each) in 220 µl of 100 mM
KCl, 20 mM triethanolamine HCl, pH 7.6, was incubated at
30°C for 10 min. After cooling to 0°C, the proteins were
crosslinked with 18 mM 2-iminothiolane as described by
Kenny et al. (38). The crosslinked sample was dialyzed against
a solution containing 20 mM sodium phosphate, pH 6.5, 7.5 M
urea, 100 mM LiCl, 5 mM iodoacetamide and loaded onto a
column of DEAE-5PW (Tosoh) equilibrated with the same
solution without iodoacetamide. Proteins were separated with
a linear gradient of 100–300 mM LiCl (see Fig. 2A). Each fraction was concentrated with Centricon YM-10 (Amicon) and
analyzed by SDS–PAGE under non-reducing and reducing
conditions.
2622 Nucleic Acids Research, 2002, Vol. 30, No. 12
Analytical ultracentrifugation
Sedimentation equilibrium studies were performed with a
Beckman Optima XL-I analytical ultracentrifuge using a
double-sector centerpiece and sapphire windows, at three rotor
speeds (12 000, 15 000 and 18 000 r.p.m.) and at 20°C. The
sample was prepared as mentioned above. Absorbance scans at
280 nm were measured in the radial step mode at 0.001 cm
intervals and data were collected taking the average of 16
measurements at each radial distance. Approach to equilibrium
was considered to be complete when replicate scans separated
by ≥6 h were indistinguishable. The partial specific volume of
the protein was assumed to be 0.73 ml/g and the density of the
solvent was assumed to be 1.00 g/ml. Analysis of the data was
carried out utilizing the program Origin 4.1.
RESULTS
In vitro interaction between isolated silkworm P1 and P2
Animal ribosomes contain two kinds of the L7/L12-like acidic
proteins, P1 and P2, unlike procaryotic ribosomes. We purified
these proteins from ribosomes of the silkworm B.mori and, in
addition, P0, corresponding to procaryotic L10. The identities of these proteins were confirmed by reactivity with
monoclonal anti-P antibody (data not shown) and partial
amino acid sequencing. The N-terminal sequence of P2 was
MRYVAAYLLAVLGGKTTPAA, which is 70% identical to rat
P2 (19) and 85% identical to Drosophila melanogaster (39). For
P1, we used a peptide produced by V8 protease digestion (40),
since the N-terminus of P1 was blocked, as in yeast (41). The
sequence was LACVYSALIL, which is 90% identical to
residues 7–16 of rat P1 (19) and 70% identical to the same
residues of D.melanogaster (42). Isolated P0, P1 and P2 were
soluble in a solution containing 0.3 M KCl in the absence of urea.
Isolated P1 and P2 showed characteristic mobilities in native
PAGE (Fig. 1A, lanes 1 and 2). A smearing of the P1 sample
(lane 1) may be due to instability of this protein under these
electrophoresis conditions. By mixing P1 with P2, a new
protein band appeared (lane 3) not detected in isolated P1 and
P2. Unexpectedly, the gel mobility of the new band was faster
than that of isolated P1 and P2 and its mobility did not change
regardless of different molar ratios of added P1 and P2 (Fig. 1B).
The new band was cut out of the gel and protein constituents
were analyzed by SDS–PAGE, followed by immunoblotting
using an anti-P monoclonal antibody that reacts with all of P0,
P1 and P2 (Fig. 1C). This band contained P1 and P2 (lane 3),
suggesting the formation of a complex composed of P1 and P2.
Relative intensity between immunostained P1 and P2 components
of the complex formed in vitro was apparently comparable with
that of the proteins in intact ribosomes (lane 4). To confirm
formation of a P1–P2 heterodimer, chemical crosslinking was
performed. The P1/P2 mixture crosslinked with 2-iminothiolane
was fractionated by ion exchange HPLC (Fig. 2A). Each fraction
was analyzed by SDS–PAGE under non-reducing conditions
(Fig. 2B). A crosslinked protein complex suggesting a dimer
(28 kDa) was detected only in fraction H (lane 8). Crosslinked
oligomers suggesting a trimer or tetramer were not formed. On
reducing the 28 kDa complex (Fig. 2C), it separated into two
protein components (lane 3) corresponding to P1 (lane 2) and
Figure 1. Formation of the P1–P2 protein complex. (A) Purified P1 (430 pmol,
lane 1) and P2 (430 pmol, lane 2) and their mixture (lane 3) in 10 µl of solution
were analyzed by 6% native PAGE, as described in Materials and Methods.
The P0.P1/P2 complex formed in the presence of P0 (Fig. 3) is also shown in
lane 4 as a comparison. (B) P2 (430 pmol) was incubated with increasing
amounts of P1: 0 (lane 1), 430 (lane 2), 860 (lane 3) and 1300 pmol (lane 4) in
10 µ l of solution. Lane 5, 860 pmol P1 alone. The samples were analyzed with
the same 6% polyacrylamide gel. (A and B) The gels were stained with
Coomassie Brilliant Blue. A new band which appeared by P1/P2 mixing and
was used for further immunoblotting analysis is arrowed. Bands for free P1
and P2 are also indicated. (C) Aliquots of 17 pmol each P1 (lane 1) and P2
(lane 2), a piece of acrylamide gel containing the newly appeared band and
2 pmol silkworm 80S ribosomes (lane 4) were subjected to SDS– PAGE,
followed by immunoblotting with anti-P monoclonal antibody (35), as
described in Materials and Methods. Appearance of a slight amount of an ∼28 kDa
component below the position of P0 in lane 3 is not reproducible. Its origin is
unknown. Considering the crosslinking results (Fig. 2), it may be a P1–P2
heterodimer tightly fixed even after SDS treatment of the gel containing the protein
complex.
P2 (lane 1), detected by immunoblotting analysis. No formation
of either P1 or P2 homodimer was detected in this experiment.
Binding of P1–P2 heterodimer to P0
We then investigated complex formation with isolated silkworm P0, P1 and P2 using native PAGE (Fig. 3A). Although
isolated P0 (lane 1) did not run in the gel under the conditions
used, complex formation was deducible from the appearance
of a new complex band or disappearance of the bands for free
P1 (lane 2) and P2 (lane 3). No complex formation was
detected in the absence of either P2 (lane 4) or P1 (lane 5). In
the presence of both P1 and P2, a stable complex was formed
(lane 6). The constituents P0, P1 and P2 of this complex were
confirmed by cutting out the band from the gel and analysis by
SDS–PAGE, followed by immunoblotting with anti-P antibody
(Fig. 3B). The results suggest that P1–P2 interaction is
required for both the proteins to assemble tightly on P0.
Nucleic Acids Research, 2002, Vol. 30, No. 12 2623
Figure 4. Sedimentation equilibrium analysis of P0.P1/P2 complex. A 0.685 mg/ml
(8.5 µM) sample of P0.P1/P2 complex was run at 20°C in 100 mM KCl, 0.2 mM
dithiothreitol, 20 mM Tris–HCl, pH 7.6. The data were collected at a rotor speed
of 12 000 r.p.m.
Figure 2. Crosslinking between P1 and P2 with 2-iminothiolane. (A) P1 and P2
proteins premixed and crosslinked with 2-iminothiolane were separated into fractions (A–H) using a DEAE-5PW column. (B) A portion of each fraction was analyzed by SDS–PAGE under non-reducing conditions as described in Materials and
Methods. Lanes 1–8 correspond to samples of fractions A–H, respectively.
(C) Protein samples from fractions C (lane 1), G (lane 2), H (lane 3) and 1.4 pmol
silkworm ribosome (lane 4) were subjected to SDS–PAGE under reducing conditions, followed by immunoblotting using anti-P monoclonal antibody.
Figure 3. Binding of P1 and P2 proteins to P0 protein. (A) Purified P0 (145 pmol,
lane 1), P1 (430 pmol, lane 2) and P2 (430 pmol, lane 3), as well as the mixtures
P0 (145 pmol) + P1 (430 pmol) (lane 4), P0 (145 pmol) + P2 (430 pmol) (lane 5)
and P0 (145 pmol) + P1 (430 pmol) + P2 (430 pmol) (lane 6), in 10 µl solutions of
20 mM Tris–HCl, pH 7.6, 0.3 M KCl, 5 mM 2-mercaptoethanol were analyzed by
6% PAGE. The protein complex band used for immunoblotting analysis in (B) is
arrowed. (B) The band of protein complex in (A) (lane 6) was cut out of the gel and
subjected to SDS–PAGE, followed by immunoblotting with anti-P antibody.
Although the complex composed of P0, P1 and P2 has been
reconstituted in vitro with proteins from human (21) and rat
(27,32), the stoichiometry of the constituents has not yet been
established.
From sedimentation equilibrium experiments, a weightaverage molecular weight is estimated by the following equation (43).
Mapp =[2RT/(1 – v ρ)ω2](dlnc/dr2)
1
where r is the radius, c is the concentration of the sample, v is
the partial specific volume of the sample, ρ is the density of the
solvent, ω is the angular velocity of the rotor (in radians/s), R
is the universal gas constant, T is the absolute temperature and
Mapp is the apparent molecular weight.
Thus, the absorbance at a specified wavelength and position
in the solution column should be given by
A(r) = A(r0)exp[MappH(r2 – r02)]
2
where A(r) represents the absorbance at radius r and A(r0) is the
absorbance at r0, the radius at the meniscus, and
H = [(1 – v ρ)ω2]/2RT
3
Figure 4 shows the results of the sedimentation equilibrium
experiments on the P0.P1/P2 complex at 12 000 r.p.m. Considering the facts that the P0.P1/P2 complex was prepared as a
fraction with a single symmetrical peak on the gel filtration
(data not shown) and that the complex gave a single band in
native PAGE, we assume the P0.P1/P2 complex to be a single
species. Figure 4 shows the non-linear least squares fitting with
this assumption. The symmetrical residuals and the small
range of 95% confidence intervals as shown in Figure 4
support this assumption. The non-linear least squares fitting
with equation 2 gave the apparent molecular weight as 7.80 ±
0.39 × 10 4. All the results with various rotor speeds (12 000,
15 000 and 18 000 r.p.m.) gave similar values within the range
of experimental error, showing that the further association of
solute is not detectable in this range of protein concentrations.
The apparent molecular weight is very close to the expected
value (80 126) of a complex of P0:P1:P2 (1:2:2) considering the
molecular weights of P0 (34 148), P1 (11 451) and P2 (11 538).
The results from these sedimentation experiments, together
with data from gel analyses (Figs 1–3), strongly suggest that
the reconstituted P0.P1/P2 complex is composed of two P1–P2
heterodimers and a monomeric P0, i.e. P0(P1–P2)2.
2624 Nucleic Acids Research, 2002, Vol. 30, No. 12
Figure 5. Binding of P0, P1 and P2 protein mixtures to GTPase-associated
RNA domain. The 32P-labeled RNA fragment (5 pmol) containing residues
1030–1127 (E.coli numbering) of B.mori wild-type 28S rRNA (A) and its
U1094/A1098 variant (B) were incubated in 10 µl of a solution without protein
(lane 1) or with a mixture of P1 and P2 (87 pmol each, lane 2), P0 (29 pmol,
lane 3) and the mixtures P0 (29 pmol) + P1 (87 pmol) (lane 4), P0 (29 pmol) + P2
(87 pmol) (lane 5) and P0 (29 pmol) + P1 (87 pmol) + P2 (87 pmol) (lane 6). The
samples were analyzed by gel retardation, as described in Materials and Methods.
(C) The B.mori U1094/A1098 variant (5 pmol) was incubated in 10 µl of a solution
with increasing amounts of isolated P0: 0 (lane 1), 29 (lane 2), 58 (lane 3) and
120 pmol (lane 4). The samples were analyzed by the same gel retardation
assay as in (B).
RNA binding of P0.P1/P2 complex
It has been shown that the P0.P1/P2 complex binds to the
GTPase-associated domain of 28S rRNA, corresponding to
residues 1030–1127 of E.coli 23S rRNA (27), presumably
through the P0 moiety. To examine the importance of P1 and
P2 in RNA binding, the experiment was performed in the
absence of either of these proteins using the silkworm RNA
fragment (Fig. 5A). Both P1 and P2, as well as P0, were
required for binding to the RNA (lane 6). No RNA binding was
detected with P1–P2 heterodimer (lane 2), P0 alone (lane 3), the
P0–P1 pair (lane 4) or the P0–P2 pair (lane 5). This experiment
was repeated using the U 1094/A 1098 RNA variant, instead of the
wild-type C1094/G1098 RNA, as RNA probe, because the structure
of the silkworm wild-type RNA is labile in solution and its
protein-binding ability is low compared with the U1094/A1098
RNA variant (34). The same results were obtained (Fig. 5B),
except that only very weak binding was detected for the P0–P2
pair (lane 5) using the RNA variant. To confirm that P0, not
P1/P2, binds directly to the RNA, a large excess of isolated P0
was added to the U 1094/A1098 RNA. A faint complex band
appeared when 24-fold P0 was added to the RNA (Fig. 5C),
although a P1/P2 mixture of the same amounts showed no
binding (data not shown). A stable RNA–protein complex was
observed only in the presence of all of P0, P1 and P2, suggesting
that the P1–P2 heterodimers greatly increased the binding
affinity of P0 to the RNA.
Figure 6. Cross-binding of silkworm P0.P1/P2 complex and eL12 to the
E.coli GTPase-associated RNA domain. 32P-labeled RNA fragments (5 pmol)
corresponding to the E.coli GTPase-associated RNA domain were incubated in 10 µ l of a solution without protein (lane 1) or with eL12 (23 pmol,
lane 2), P0.P1/P2 complex (37 pmol, lane 3) and their mixture (lane 4). The
samples were analyzed by gel retardation, as described in Materials and
Methods.
and eEF-2 has been established (28). We attempted to use this
hybrid system to study the function of the silkworm ribosomal
proteins. For this purpose, the prepared silkworm samples
must cross-bind to the E.coli GTPase-associated RNA domain.
As shown in Figure 6, silkworm P0.P1/P2 (lane 3) and L11-like
protein eL12 (lane 2) as well as both the proteins (lane 4)
bound strongly to the E.coli RNA.
The silkworm protein samples were added to the core E.coli
ribosomes deficient in L10.L7/L12 and L11 (28) and tested for
function (Fig. 7). The hybrid ribosomes containing silkworm
P0.P1/P2 and eL12 showed eucaryotic eEF-2-dependent
GTPase activity (Fig. 7A) and polyphenylalanine synthesis
(Fig. 7B). These activities were comparable with those of the
previous hybrid sample containing rat ribosomal proteins (28).
Removing either P1 or P2 markedly reduced both the activities. In
the absence of P1, addition of P0/P2 in 5-fold excess to the
E.coli 50S subunit core gave no effect on the GTPase activity
(Fig. 7A). Likewise, addition of excess amounts of P0/P1 did
not recover the activity in the absence of P2. In the absence of
P0, addition of P1/P2 gave no stimulation of the activity (data
not shown).
To test whether the activity of the hybrid ribosome is due to
binding of the silkworm proteins to the GTPase-associated
RNA domain within the E.coli ribosome, a competition study
was performed using the E.coli RNA fragment used in the
binding assay (Fig. 6) as a competitor. On addition of the RNA
competitor, polyphenylalanine synthetic activity of the
ribosome sample was reduced to ∼35% of the original activity
(Fig. 7C). Addition of RNA itself showed no effect on
polyphenylalanine synthesis by the intact silkworm ribosomes.
The results indicate that the activity of the hybrid ribosomes
was caused by interaction between the silkworm ribosomal
proteins and the E.coli GTPase-associated RNA domain.
Functional properties of P0.P1/P2 complex
DISCUSSION
It is important to evaluate the present in vitro binding data in
the aspect of ribosome function. To test the function of P0, P1
and P2 proteins, we recently developed a useful hybrid
system in which E.coli L10.L7/L12 and L11 bound to the
GTPase-associated domain of 23S rRNA were replaced with rat
P0.P1/P2 complex and eL12 on the 50S subunit. Accessibility of
the hybrid ribosome to eucaryotic elongation factors eEF-1α
The structure of the ribosomal GTPase center within the large
subunit is not resolved well by the current X-ray crystallography (44,45), probably because of its flexible nature. Many
lines of biochemical evidence, however, indicate that the
pentameric acidic protein complex L10(L7/L12)2(L7/L12) 2
binds to the GTPase-associated domain of 23S rRNA and
constitutes a major part of the functional center in procaryotic
Nucleic Acids Research, 2002, Vol. 30, No. 12 2625
Figure 7. Functional assays of the P0, P1 and P2 samples in a hybrid system
with E.coli ribosomes. (A) Escherichia coli 50S cores deficient in L10.L7/L12
complex and L11 (2.5 pmol) were preincubated in 10 µl of a solution with
increasing amounts of the P0.P1/P2 complex (circles), P0/P1 mixture
(diamonds), P0/P2 mixture (triangles) and P0 alone (squares), together with
5.7 pmol eL12. The ribosome samples were tested for GTPase activity dependent
on eucaryotic eEF-2 purified from pig liver (50), as described in Materials and
Methods. (B) Escherichia coli 50S cores (10 pmol) were preincubated in 25 µl
of a solution without any P proteins (bar 1) or with 20 pmol P0 (bar 2), P0
(20 pmol) + P1 (61 pmol) (bar 3), P0 (20 pmol) + P2 (61 pmol) (bar 4) and P0
(20 pmol) + P1 (61 pmol) + P2 (61 pmol) (bar 5), together with 23 pmol eL12.
The ribosome samples were tested for poly(U)-dependent polyphenylalanine
synthesis by adding 800 µg S200 fraction as a source of silkworm elongation
factors, as described in Materials and Methods. The activity was also assayed
with 10 pmol silkworm 80S ribosomes under the same conditions except that
the salt concentrations used were 5 mM MgCl2, 50 mM NH4Cl, 100 mM KCl,
50 mM Tris–HCl, pH 7.6, 0.2 mM dithiothreitol (bar 6). (C) The E.coli 50S
cores (10 pmol) were preincubated in 25 µl of a solution with P0.P1/P2
complex (20 pmol) and eL12 (23 pmol) in the presence of increasing amounts
of the RNA fragment corresponding to the E.coli GTPase-associated domain
(circles). The intact silkworm 80S ribosomes (10 pmol) were also incubated
with the same RNA fragments (squares). The samples were assayed for
polyphenylalanine synthetic activity, as shown in (B).
ribosomes. The eucaryotic proteins P0 and P1/P2 are the counterparts of procaryotic L10 and L7/L12, respectively. We here
investigated interactions among silkworm P0, P1 and P2 by
using the isolated proteins and showed that a P0:P1:P2 (molar
ratio 1:2:2) pentameric complex was reconstituted in vitro,
which appears to be a functional unit activating ribosomes by
its binding to the GTPase-associated RNA domain. A major
difference between the eucaryotic and procaryotic acidic
proteins is that there are two types, P1 and P2 (three types in
some plants; 46), of the proteins in eucaryotes that are
expressed from different genes (19,21,24). The present data
clearly confirmed the formation of a P1–P2 heterodimer in vitro
(Figs 1 and 2). The P1–P2 heterodimer seems to be compact
and stable, because the band of the dimer is distinct on native
electrophoretic gels and its mobility is higher than isolated P1
and P2 (Fig. 1A). Formation of the heterodimer is consistent
with previous data from different approaches (26,29,30,31,33).
All of the present results lead to the conclusion that formation
of the P1–P2 heterodimer is a key step in assembly of the
P0(P1–P)2 pentameric complex and its rRNA binding to
constitute the silkworm ribosomal GTPase center.
Binding of P0 to the GTPase-associated RNA domain
appears to be the most crucial step linked directly to ribosome
function. It is likely that the RNA-binding site lies in the
N-terminal half of P0 protein (47,48) and the P1/P2-binding
site in the C-terminal region (24) (Fig. 8). However, it has been
very hard to investigate the molecular details of the RNAbinding mechanism of P0, because of its instability in the
isolated state. Although rat P0 protein has been prepared from
ribosomes (27) and by overexpression in E.coli cells (32), the
isolated protein samples are insoluble without P1 and P2. In
the present studies, we could isolate P0 protein in a soluble
state from silkworm ribosomes. The amino acid sequence of
silkworm P0 shows 69% identity with rat P0 (Fig. 8). One or
some of the divergent parts of the molecule seem to contribute
to the solubility of silkworm P0. From the fact that the very
low RNA-binding ability of isolated P0 is significantly
increased by addition of both P1 and P2 (Fig. 5), it is conceivable that the conformation of the RNA-binding site of P0
apparently changes on binding of P1–P2 heterodimers to the
C-terminal side of the molecule. This allosteric conformational
change induced by P1–P2 heterodimers may be important for
RNA binding and ribosome function. We propose here that the
P1–P2 heterodimer, but neither P1 nor P2 alone, plays a role in
modulating the structure and function of P0 protein at least in
the case of silkworm proteins.
There is a major difference between the present study and
that of yeast mutants. The yeast mutant deficient in P1 and P2
was made by disruption of genes for both proteins (for P1α,
P1β, P2α and P2β) (24). This P1/P2-deficient yeast ribosome
retained P0 protein and showed a reduced level of polyphenylalanine synthetic activity. Growth of this strain was 3-fold
slower compared with the wild-type (24). In contrast, our results
indicate that isolated silkworm P0 hardly binds to the GTPaseassociated RNA domain without P1 and P2 and does not activate
the ribosome. One explanation for this discrepancy may be the
difference in experimental conditions between in vitro and in vivo
studies. In yeast cells, there may be components compensating
for the P1–P2 modulation of P0. A certain ribosomal protein
other than P1/P2 may play such a role. A second possible
explanation is species differences. Unlike animal P0, yeast P0
2626 Nucleic Acids Research, 2002, Vol. 30, No. 12
Figure 8. Comparison of amino acid sequence for silkworm (Bmo) P0 with those for the rat (Rno) and yeast (Sce) homologs. The sequence of silkworm P0 was
derived by assembling several partial sequence data (http://www.ab.a.u-tokyo.ac.jp/silkbase/) of the B.mori cDNA library (51). The conserved arginine-rich region
(positions 44–67), which is most likely to participate in RNA binding (47), and the P1/P2-binding region (positions 182–293) (24) are indicated.
may bind to the RNA without the help of other proteins,
although this has not yet been tested. Modes of interaction
between P0 and P1/P2 and their functional significance may
have diverged during evolution. In fact, identity of amino acid
sequence around the P1/P2-binding region (28.6% identity) on
P0 is lower than that of full-length P0 (44.2%) as well as the
N-terminal arginine-rich region (62.5% identity) among yeast,
silkworm and rat (Fig. 8).
Some possible models of protein topography of P0, P1 and
P2 on animal ribosomes have been presented (32): (i) P1–P1
and P2–P2 homodimers bind separately to P0; (ii) two P1–P2
heterodimers bind to P0 so that the two dimers come close to
each other; (iii) as (ii), but the two dimers do not come close to
each other. Our present results strengthen the idea of
heterodimer formation and support model (ii) or (iii). The two
E.coli L7/L12 homodimers are known to bind very close to
each other on the C-terminus of L10 (17); if the same is true in
animal ribosomes, then model (ii) may be the actual molecular
arrangement. Previous P1–P1 and P2–P2 crosslinking with
2-iminothiolane (22) may be explained by model (ii).
Eucaryotic translation is highly regulated. For instance, the
GTPase turnover by eucaryotic 80S ribosomes and the translocase
eEF-2 is 10-fold slower than that by procaryotic 70S ribosomes
and EF-G (49). Our previous study demonstrated that the
P0.P1/P2 complex participates in the eucaryotic characteristics
(49). The presence of two kinds of the acidic proteins and their
interaction may be involved in the characteristics. Strong
binding of P0.P1/P2 complex to the GTPase-associated RNA
domain, compared with the E.coli equivalent L10.L7/L12
complex (49), may be also related to the eucaryotic-specific
properties in the translation process.
ACKNOWLEDGEMENTS
We thank K. Mita (National Institute of Radiological Sciences,
Japan) for providing sequence data of silkworm ribosomal
proteins and S. Uchiyama (Osaka University, Japan) for
helpful discussion on equilibrium sedimentation. We also
thank Gene Research Center Shinshu University for giving full
facilities for the research. This work was supported by grantsin-aid for scientific research (12660053 and 13033015) and for
COE research (10CE2003) from the Ministry of Education,
Science, Sports and Culture of Japan.
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