From the Graduate School of Pharmaceutical Sciences,
Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan, the
§ Research Institute for Microbial Diseases, Osaka
University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan, the
Membrane Dynamics Research Group, RIKEN, Harima Institute at
SPring-8, 1-1-1 Koto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, and the ** Graduate School of Pharmaceutical Sciences,
Kumamoto University, 5-1 Oe-honmachi,
Kumamoto 862-0973, Japan
Received for publication, August 8, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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X-ray and NMR analyses on ribosome
recycling factors (RRFs) from thermophilic bacteria showed that they
display a tRNA-like L-shaped conformation consisting of two domains.
Since then, it has been accepted that domain I, consisting of a
three-helix bundle, corresponds to the anticodon arm of tRNA and domain
II and a At the final step in each cycle of protein biosynthesis, ribosome
recycling factor (RRF)1
disassembles the post-termination complex, which is composed of 70 S
ribosome, deacylated tRNA and mRNA in concert with elongation factor G (EF-G) in a GTP-dependent reaction (1-3). In
Escherichia coli, the lack of RRF causes increased errors in
translation (4) and unscheduled reinitiations (5, 6).
Presently there are four published reports regarding the
three-dimensional structures of RRF molecules. They are crystal
structures of RRFs from Thermotoga maritima (7),
Escherichia coli (8), and Thermus thermophilus
(9) and a solution structure of RRF from Aquifex
aeolicus (10). All of these structures consist of two domains;
domain I displays a three-helix bundle structure, and domain II exists
as a three layer However, the above mentioned concept of molecular mimicry presents a
problem. RRFs whose structures have been determined to be a strict
L-shape, were obtained from thermophilic bacteria and did not show
polysome breakdown activity in vitro system containing EF-G
and puromycin-treated polysome fraction from E. coli
(11-13). RRFs that demonstrated activity in that system were only
mesophilic bacteria, i.e. E. coli and
Pseudomonas aeruginosa (14). It has been also demonstrated
in vivo by Janosi et al. using
temperature-sensitive mutant of E. coli that RRFs from
mesophilic bacteria exhibit an RRF activity but RRFs from thermophilic
bacteria do not (15).
To clarify the problem, we would like to know if the unique structure
of E. coli RRF is common in mesophilic bacteria and essential for RRF activity, or if the unique structure is just an
exception because of some artifact like detergent binding. Actually,
the detergent molecule, which was required to crystallize the protein,
was reported to attach to the hinge region connecting the two domains
of E. coli RRF (16).
Here, we obtained a crystal of RRF from Vibrio
parahaemolyticus that was free of detergent. This is a mesophilic
bacterium that is a relative of E. coli, and its RRF was
expected to show activity in the E. coli system. The
molecular cloning and characterization will be described, and the
crystal structure of this RRF at 2.2 Å resolution will be described as
well. To obtain deeper insight into the mechanism of disassembly of the
termination complex, we prepared the domain I portion of RRF without
domain II and investigated the interaction between domain I and
ribosome. Here we propose a new binding mode between ribosome and
RRF.
Bacterial Strains, Plasmids, and Culture
Conditions--
V. parahaemolyticus strain KXV237(O3:K6)
(17) was used for molecular cloning. E. coli strain DH5 Molecular Cloning--
To select V. parahaemolyticus
RRF DNA (frr) clones, a partial E. coli
frr fragment was used as a DNA probe. The DIG system (Roche
Molecular Biochemicals) was used for labeling of the DNA probe.
Primers (5'-CATGGACAAATGCGTAGAAGCG-3' and 5'-CTTCTTTGTCTGCCAGCGCC-3') for PCR were designed to amplify the DNA fragment encoding residues Met-13-Glu-179 of E. coli RRF. The pET22b(+) plasmid
(Novagen) carrying E. coli frr was used as a PCR template.
NotI fragments of genome DNA of V. parahaemolyticus KXV237 were hybridized with the probe. Pulse
field gel electrophoresis and subsequent Southern hybridization methods
have been applied as described previously (18). The luminescent
fragment of ~168 kbp was collected and digested with PstI.
Digested products were subjected to electrophoresis on agarose gel
(0.8%) and hybridized again. The PstI fragment of 4.1 kbp
was collected and inserted into PstI site of pBluescript II
KS(
Restriction map of the 4.1-kbp PstI fragment was created
with conventional methods (19). The fragment of 1.2 kbp between SpeI and HindIII sites included the
frr gene. The fragment was reconstructed into pBluescriptII
KS( Construction of RRF-DI (E. coli Domain I)--
To elucidate the
role of domain I in RRF, we constructed an expression vector coding a
recombinant protein with the Met-1-Gly 30 and Thr-106-Phe 185 segments of E. coli RRF that correspond to domain I. The two
segments were connected by a flexible linker composed of glycine
residues. In our preliminary result of E. coli R132G mutant,
the distance between Gly-30 and Thr-106 was 9.8 Å (21). Thus three
glycine residues were sufficient to connect the two segments. The
expression vector, (1-30)-Gly-Gly-Gly-(106-185), was synthesized by
PCR using the plasmid pET22b(+) containing E. coli RRF as a
template. Oligonucleotides 5'-ACCACCACCCGTGCGTATTTTGCTGATTTGGGTTTTG-3' and 5'-GGTACGGAAGAACGTCGTAAAGATCTGACCAAAATC-3' were the primers. The
linked domain I was named as RRF-DI. Confirmation of the DNA sequence
was carried out before the protein expression.
Protein Expression and Purification--
The DNA fragment of
V. parahaemolyticus encoding RRF sequence was amplified by
PCR with pBluescriptII KS(
The expression and purification of RRF-DI were performed as follows.
E. coli strain BL21(DE3) was transformed with the plasmid carrying RRF-DI. Transformants were grown first in 5 ml, then in 1 liter of LB medium containing 100 µg/ml ampicillin at 37 °C. When
the absorbance of the culture reached 0.6 at 660 nm, isopropyl-1-thio- Circular Dichroism (CD) Measurement--
Circular dichroism
spectra of E. coli RRF and RRF-DI were recorded on an AVIV
model 202 spectrophotometer (AVIV) with a 1-mm path-length cuvette at
10 °C using 5 µM protein solutions in a buffer (20 mM sodium acetate, pH 3.6, and 50 mM NaCl).
The Functional Assessment of RRFs--
Purified recombinant
V. parahaemolyticus RRF, E. coli RRF, and RRF-DI
were assayed with puromycin-treated polysome as a substrate (11). The
polysome fraction was prepared from E. coli Q13 strain and
used for the post-termination complex disassembly reaction by RRF as
described previously (22). The reaction mixture (150 µl) contained 10 mM Tris-HCl at pH 7.4, 8.2 mM
MgCl2, 80 mM NH4Cl, 1 mM DTT, 0.16 mM GTP, 0.05 mM
puromycin, 1.3 A260 units of polysomes, 1.6 µM EF-G, and various amounts of RRF. The mixture was
incubated at 30 °C for 20 min, followed by separation of the mixture
into polysome and monosomes with 5-45% sucrose density gradient
ultracentrifugation (250,000 × g, 45 min at 4 °C).
Quantitative measurements of dissociation products were determined from
the absorbance at 260 nm with a Piston Gradient Fractionater (Biocomp
Inc.). The inhibitory effect of RRF-DI on the recycling activity of
E. coli RRF was estimated by the addition of an excess
amount of RRF-DI in the presence of 1 µM E. coli RRF.
Crystallization and Data Collection--
The V. parahaemolyticus RRF crystals were grown at 4 °C using the
hanging drop vapor diffusion method. Drops consist of 2 µl of protein
solution (10 mg/ml) with 2 µl of reservoir solution containing 50 mM MES-NaOH at pH 6.2, 200 mM sodium acetate,
and 23-25% w/v PEG 8000. During crystallization, fusidic acid
and GTP were added to the drop to a final concentration of 0.75 and 5 mM, respectively. This condition produced several crystals
within 1 day, and crystals reached full size (typically 0.2 × 0.2 × 0.2 mm) within 3 days. The crystal was transferred into a
cryoprotectant consisting of 50 mM MES-NaOH at pH 6.2, 200 mM sodium acetate, 25% w/v PEG 8000, 15% v/v
2-methyl-2,4-pentanediol, and 5% v/v 2-propanol and flash frozen in
liquid nitrogen. The x-ray diffraction data set at 2.2 Å resolution
were collected using synchrotron radiation with a crystal-to-detector
distance of 430 mm at station BL18B of the Photon Factory in the High
Energy Accelerator Research Organization (Tsukuba, Japan). Data frames
were collected on a Weissenberg camera with an imaging plate for
macromolecular crystallography (23). The data were processed using the
DENZO and SCALEPACK programs of the HKL package (24). The crystal of
the V. parahaemolyticus RRF belongs to the trigonal space
group R32 with unit-cell parameters a = b = 84.80 and c = 142.73 Å. Assuming
one molecule per asymmetric unit, the calculated Matthews coefficient
VM value is 2.40 Å3/Da (25). The
solvent content of the crystal was therefore calculated to be 48.8%.
Data collection statistics are given in Table I.
Structure Determination and Refinement--
Molecular
replacement calculations were performed on the V. parahaemolyticus RRF using the program AMoRe in the CCP4 suite (26). As the search model, we used the crystal structure of E. coli RRF mutant, R132G, which has been reported elsewhere (21). In
generating the search model, the residues that are not identical between sequences were replaced by Ala residues. As a result, a single
solution was found with a correlation coefficient of 0.316 and
R-factor of 55.2% (8.0-4.0 Å) after the
translation-function calculation. To improve the accuracy of the
solution, the resultant structure was subjected to 40 cycles of rigid
body refinement using data from 8.0 to 3.0 Å resolution. The
R-factor was refined to 46.2%.
Structure refinement was carried out further using the programs X-PLOR
(27), CNS (28), and TOM (29). After a number of cycles of model fitting
and refinement, it was possible to trace almost the entire molecule. In
this step, the alanine-replaced residues were restored to the original
amino acid residues. Later refinement steps included the refinement of
grouped or individual temperature factors. Omit maps were used to check
the model. Water molecules were identified using waterpick procedure in
the CNS program.
The final model included all 185 amino acids and 227 water molecules.
Under crystallization conditions, the solution contains fusidic
acid and GTP. Although these compounds are considered essential for
crystallization of V. parahaemolyticus RRF, only protein and
water molecules were observed in electron density maps. The final
R-factor and Rfree were 20.3% and
27.3%, respectively, with a mean positional error of 0.241 Å (30).
This model had excellent stereochemistry when it was evaluated with the
program PROCHECK (31). On the Ramachandran plot, 94.7% of the
non-glycine residues are in the most favored region, and none of the
residues lie in the generously allowed and disallowed regions. Root
mean square deviations (r.m.s.d.) of bond lengths and angles are
0.005 Å and 1.078 °, respectively. Table
I summarizes the refinement statistics.
The final coordinates and structure factor have been deposited in the
Protein Data Bank (PDB number 1IS1).
Ribosome Binding Assay--
Surface plasmon resonance
experiments were carried out on a Biacore 2000 biosensor system
(Biacore AB) to qualitatively examine the binding of 70 S ribosome or
its subunits to RRF at 25 °C. Immobilization of RRF on a CM5 sensor
chip (Biacore) was carried out according to the manufacturer's
standard protocol using an amine coupling method, by which the protein
is covalently immobilized with N-hydroxysuccinimide-based
chemistry via lysine
For monitoring the interaction, all procedures were automated to create
repetitive cycling of sample injection and regeneration. Ribosomes,
over a range of concentrations between 60 and 900 nM, were
injected simultaneously into cells 1 and 2. Then the response from cell
1 was corrected by subtracting the response from cell 2, which arose
from nonspecific interactions between ribosomes and the dextran matrix
on the sensor chip. The HEPES buffer (10 mM HEPES-NaOH, pH
7.4, 50 mM NH4Cl, 8.2 mM
Mg(OAc)2, 1 mM DTT, and 0.005% Tween 20) was
used for sample dilution and running buffer at a flow rate of 10 µl/min.
To investigate inhibition of wild-type RRF binding to the 50 S subunit
by RRF-DI, the 50 S subunit was preincubated with various concentrations of RRF-DI (0.05-3 µM). Then the mixture
was injected on a CM5 sensor chip. In this experiment, wild-type
E. coli RRF was immobilized on the sensor chip using the
above procedure.
Binding of RRF-DI to 70 S ribosome or its subunits was further measured
by a filtering technique. RRF-DI was incubated with 0.25 µM ribosome in 40 µl of Tris buffer (10 mM
Tris-HCl, pH 7.4, 8.2 mM MgSO4, 50 mM NH4Cl, 1 mM DTT, and 0.3 mM EDTA) at 30 °C for 10 min. The mixture was applied
onto Microcon YM-100 (Millipore) and centrifuged for 5 min at
3,000 × g to trap the ribosome-bound RRF-DI. 40 µl
of the same buffer was loaded into the column and centrifuged for 10 min at 3,000 × g to wash out the unbound RRF-DI. The
washing was performed two times. Then the filter was incubated with 40 µl of the buffer for 1 min at room temperature. The ribosome-bound RRF-DI was collected from the inverted column by centrifugation. The
recovered RRF-DI was detected by Western blotting with anti E. coli RRF rabbit antibody (1:10,000 dilution). Bound RRF-DI was
quantified by the blotting of known amounts of RRF-DI standards. To
quantify nonspecific binding between RRF-DI and filter apparatus, control experiments without ribosomes were done. Binding of wild-type RRF to 70 S ribosome or its subunits was measured in the same way.
Nucleotide Sequence of the frr Gene and Comparison of Its Amino
Acid Sequence with Other RRFs--
The 1.2 kbp
SpeI-HindIII fragment containing V. parahaemolyticus frr was sequenced. The fragment had only one
major open reading frame. Pribnow box,
The amino acid sequence of V. parahaemolyticus RRF was
compared with other previously reported RRF sequences (Fig.
1). Most RRFs contain 185 amino acids.
The amino acid residues conserved in other species are also conserved
in V. parahaemolyticus RRF. All reported RRFs are highly
homologous (e.g. E. coli 70.1%, P. aeruginosa 63.2%, Staphylococcus aureus 45.4%,
T. thermophilus 42.7%). Such high level homology strongly
suggests that RRFs have similar tertiary structures.
V. parahaemolyticus RRF Is Functional in the E. coli
System--
The ribosome recycling activity of recombinant V. parahaemolyticus RRF was tested in a polysome breakdown assay. As
shown in Fig. 2, V. parahaemolyticus RRF shows polysome breakdown activity in the
present assay system containing E. coli polysomes. The fraction of polysome converted into 70 S ribosome increases as the
amount of V. parahaemolyticus RRF increases. The fraction of
converted polysome reaches a constant value of ~40% with the addition of a sufficient amount of V. parahaemolyticus RRF
(>3 µM). This converted fraction by V. parahaemolyticus RRF is somewhat smaller than, but close to, that
processed by E. coli RRF where 50% of polysome was
processed with the same amount of V. parahaemolyticus RRF.
The initial slopes of RRFs from E. coli and V. parahaemolyticus in this assay indicate that the order of specific
activity is similar between these two RRFs. In contrast, RRF from
thermophilic bacteria show no activity using this assay system (12).
Overall Architecture--
The V. parahaemolyticus RRF
structure has a strictly L-shaped conformation with two
domains and dimensions of ~60 × 51 × 25 Å (Fig.
3). The overall structure of V. parahaemolyticus RRF is similar to those of T. maritima
(7), E. coli (8), T. thermophilus (9) and
A. aeolicus (10) RRFs. Domain I (residues 1-30 and 104-185) is a three-helix bundle. Domain II (residues 31-103) is
composed of two
The number of hydrogen bonds was calculated by the Insight II program
(Accelrys Inc.) based on atomic distances. Besides the hydrogen bonds
in
The contact area between the two domains is 1066 Å2, which
corresponds to 9.6% of the total surface area (11,086 Å2)
of RRF. This small contact area and the small number of interdomain interactions suggest that the interaction between domains I and II is
relatively weak and hence the relative configuration of domains I and
II is expected to be flexible.
Comparison to Other RRFs--
As shown in Fig.
4A, the overall folds and
dimensions of domains I and II of V. parahaemolyticus RRF
are similar to those RRF structures that were previously determined by
x-ray crystallography and NMR spectroscopy. Furthermore,
during the survey of crystallizing conditions of RRFs, we succeeded in
crystallizing one RRF mutant from E. coli, R132G, that is
free of detergent. The preliminary study on this mutant has been
reported elsewhere (21). The x-ray analysis on V. parahaemolyticus RRF and this RRF mutant revealed that both RRFs
from mesophilic bacteria have a strict L-shaped structure
very close to those of the above mentioned RRFs from thermophilic
bacteria. On the other hand, spatial arrangements of domains I and II
are different among all of RRFs.
The relative orientation of these domains in RRFs for which tertiary
structures have been reported are plotted in Fig. 4B using
three spherical polar angles: Ribosome Binding of RRF-DI--
We have concluded above that the
RRF molecules consist of two domains in a strict L-shaped
structure where intramolecular movement is restricted in a plane. This
raised the question whether each domain plays a different role. To
clarify this question, we first tried to split the molecule at the
hinge region by inserting a scissile sequence for suitable proteinases,
but we failed. This may be because the inserted portion was not exposed
enough for enzymes to access it. Then we designed the recombinant
proteins corresponding to each domain. For the one corresponding to
domain II, we constructed an expression vector coding Arg-31-Leu-105, but the desired polypeptide precipitated as an inclusion body in
transformed cells. However, we succeeded in expressing a polypeptide consisting of the fragments; Met-1-Gly-30 and Thr-106-Phe-185, with
Gly-Gly-Gly, as a linker between the fragments. This polypeptide, named
RRF-DI, was characterized by CD measurements.
As shown in Fig. 5, the CD spectrum of
RRF-DI showed the characteristic profile of an
We wondered whether RRF-DI exhibits the activity of disassembly of the
post-termination complex. According to Fig.
6, RRF-DI does not show any ribosome
recycling activity. Furthermore RRF-DI inhibited wild-type RRF
activity. By mixing of RRF-DI with wild-type RRF (~50:1) in the
reaction mixture, the yield of monosome was reduced to ~30% compared
with that converted by wild-type RRF in the absence of RRF-DI. These
results indicate that both RRF-DI and wild-type RRF share the same
binding site.
Furthermore we have shown that the positively charged cluster region
around Arg-132 on domain I plays a crucial role in binding to the 50 S
subunit of ribosome (33). Consequently another question arose whether
domain I, where Arg-132 is located, interacts with the ribosome or with
the 50 S subunit.
Biacore experiments have been shown to be useful for investigations of
interactions involving ribosomes (34). To examine the interactions
between RRF and ribosomes, we carried out Biacore experiments.
Although, nonspecific interactions, which were estimated in blank cell,
were not negligible in the experiments (see supplemental Fig. S1 at
http://www.jbc.org), qualitative results were reproducible. As shown in
Fig. 7A,
resulting sensorgrams showed qualitatively that RRF-DI is bound to 70 S
ribosome and the 50 S subunit but not to the 30 S subunit. The
prohibition on the binding of the 50 S subunit to the immobilized
wild-type RRF by RRF-DI was also observed (Fig. 7B). The
response for the binding of wild-type RRF to 70 S ribosome was
relatively low. Presumably, the immobilization of wild-type RRF
efficiently reduced the accessibility for such a large particle as 70 S
ribosome. Dose-dependent responses were observed in these
experiments; however, the responses were not proportional to the
amounts of injected ribosomes. The apparent affinity might be affected
by the nonspecific interactions and the immobilization procedure. Their
effects were estimated in the blank cell and were not negligible in
these studies (supplemental Fig. S1). Because the apparent affinity
might be affected by such nonspecific interactions, the affinities
e.g. between wild-type RRF and RRF-DI cannot be
quantitatively compared with the Biacore technique.
Thus, to examine the binding affinities of wild-type RRF and RRF-DI to
ribosomes more quantitatively, we performed binding assays using a
filtering technique (Fig. 7, C and D, and Table II). Various amounts of wild-type RRF or
RRF-DI were mixed with 70 S ribosome, the 50 S subunit, or the 30 S
subunit, and then the amounts of bound RRFs were determined. In these
experiments, nonspecific binding of wild-type RRF or RRF-DI to filter
apparatus was found to be negligible (Fig. 7C). Binding of
both proteins to 70 S ribosome and the 50 S subunit was observed, but
was not observed to the 30 S subunit. Although these measured
dissociation constants may be overestimated because the filter
technique is a non-equilibrium technique, these values should be
relatively meaningful (see discussion by Hirokawa et al.
(35)). The apparent dissociation constant of RRF-DI from the 50 S
subunit was estimated to be 0.52 µM. This value is
slightly higher than that of wild-type RRF, 0.16 µM. For
70 S ribosome, the apparent dissociation constant of RRF-DI was 0.16 µM, which was slightly lower than 0.42 µM
of wild-type RRF. This similarity of dissociation constants for RRF-DI and wild-type RRF indicates that the binding modes of these molecules are likely to be comparable to each other. Our results appear to
contradict a previous report by Ishino et al. that RRF
interacts weakly with the 30 S subunit (33). This is likely due to the fact that the low ionic strength buffer (10 mM Tris-HCl, pH
7.4, 8.2 mM MgSO4, 10 mM
NH4Cl, 1 mM DTT) was used in their experiment and that at such a condition nonspecific interactions easily occur.
Structures of the ribosome and its subunits have been elucidated
by cryo-electron microscopy and x-ray crystallography (36). Recent
crystallographic studies revealed the structure of the ribosome at high
resolution and the location of ribosome-bound tRNAs (37). In the
present study, the crystal structure of V. parahaemolyticus
RRF and the binding mode of RRF to the ribosome were determined. The
resemblance of the structure of RRF to that of tRNA in terms of shape
and size has been recognized as representative of molecular mimicry
(7). To examine this mimicry we attempted to construct the RRF-ribosome
complex model by replacing tRNA in the ribosome structure with RRF.
It is generally accepted that domains I and II of RRF correspond to the
anticodon and acceptor arms of tRNA, respectively. This assumption,
which has been suggested by Selmer et al., is based on the
structural similarity between T. maritima RRF and tRNA (7).
In Selmer's model, domain I of A-site-bound RRF would point toward the
decoding area of the 30 S subunit and domain II would point toward the
peptidyl transferase region of the 50 S subunit. A cross section of the
RRF-ribosome complex derived from Selmer's model, as shown in Fig.
8B, clearly shows that domain I of RRF contacts primarily the 30 S subunit, whereas domain II contacts the 50 S subunit.
/
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sandwich structure, corresponds to the acceptor
arm. In this study, we obtained a RRF from a mesophilic bacterium,
Vibrio parahaemolyticus, by gene cloning
and carried out an x-ray analysis on it at 2.2 Å resolution. This RRF
was shown to be active in an in vitro assay system using
Escherichia coli polysomes and elongation factor G (EF-G).
In contrast, the above-mentioned RRFs from thermophilic bacteria were
inactive in such a system. Analysis of the relative orientations
between the two domains in the structures of various RRFs, including
this RRF from mesophilic bacterium, revealed that domain II rotates
about the long axis of the helix bundle of domain I. To elucidate the
ribosome binding site of RRF, the peptide fragment (RRF-DI)
corresponding to domain I of RRF was expressed and characterized.
RRF-DI is bound to 70 S ribosome and the 50 S subunit with an
affinity similar to that of wild-type RRF. But it does not bind to the
30 S subunit. These findings caused us to reinvestigate the concept of
the mimicry of RRF to tRNA and to propose a new model where domain I
corresponds to the acceptor arm of tRNA and domain II corresponds to
the anticodon arm. This is just the reverse of a model that is now
widely accepted. However, the new model is in better agreement with
published biological findings.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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sandwich structure. Except for E. coli RRF, the relative angles of these two domains are ~90 °
and thus their overall structures are arranged in an L-shaped
conformation very similar to that of tRNA. Based on this similarity, a
concept of molecular mimicry was proposed. It was suggested that
domains I and II of RRF correspond to the anticodon and acceptor arms
of tRNA, respectively (7). Thus it was proposed as a hypothetical
mechanism that RRF would be bound first to the A-site of the ribosome
and then translocated by EF-G to the P-site in a manner similar to that
of tRNA, leading to the disassembly of the post-termination complex
(7). Meanwhile the tertiary structure of E. coli RRF
determined by an x-ray analysis showed that even though the secondary
structure elements of each domain was almost the same as those of the
other RRFs, the spatial arrangement of the two domains is in an obtuse
angle so that it does not look like tRNA (8). One could explain that
the difference in the structure might be caused by a large difference
between the species of origin because E. coli is a
mesophilic bacterium but the other three are thermophilic bacteria.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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was used as a host strain for cloned plasmid DNA. E. coli
strain BL21(DE3) was used for protein expression. Luria-Bertani (LB)
broth (Nakalai tesque) was used in liquid media and solid agar media
(1.5%) for routine cultivation of bacteria. The media were
supplemented with 100 µg/ml ampicillin.
) (Toyobo).
) vector and sequenced. DNA sequencing was performed by an
automated DNA sequencer (Shimadzu) with a fluorescent dye primer
method. The DNA sequence was analyzed with the Gene Web II program
(20).
) vector as a template. The GTG start codon
in the cloned sequence was converted to ATG in PCR primer. The PCR
product was cloned into the pET22b(+) plasmid vector. The construct was
introduced into the E. coli BL21(DE3) strain. The
transformant was grown at 37 °C. When the absorbance of the culture
reached 0.6 at 660 nm, protein expression was induced by the addition
of isopropyl-1-thio-
-D-galactopyranoside to a final concentration of 1 mM. After a 3-h incubation from
the induction, cells were harvested and disrupted by sonication in
buffer A (20 mM Tris-HCl, pH 8.0, 10 mM
MgCl2, 2 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride). After the centrifugation of the homogenate at 300,000 × g for 3 h, the
supernatant was applied onto a 5-ml Hi-Trap Q column (Amersham
Biosciences) equilibrated with buffer A. The flowthrough fraction
containing RRF was dialyzed against buffer B (20 mM sodium
acetate, pH 5.0, 10 mM NH4Cl, and 6 mM 2-mercaptoethanol) and applied onto a 5-ml Hi-Trap SP
column (Amersham Biosciences) equilibrated with buffer B. The eluted fractions containing RRF were concentrated by Centricon YM-10 (Millipore), and the concentrate was further purified by a Superdex 75 pg column (Amersham Biosciences) equilibrated with buffer A containing 300 mM sodium acetate. The product homogeneity
was determined by SDS-PAGE, and the molecular weight was checked by matrix-assisted laser desorption/ionization time of flight mass spectrometry (Applied Biosystems).
-D-galactopyranoside was added to a
final concentration of 1 mM for protein expression,
followed by culturing for 3 h. Cells were harvested and disrupted
by ultrasonic treatment in buffer C (20 mM Tris-HCl, pH
7.4, and 6 mM 2-mercaptoethanol). After centrifugation of
the homogenate at 14,000 × g for 30 min, the
supernatant was applied onto a 5-ml Hi-Trap Q column (Amersham Biosciences) equilibrated with buffer C. The eluted fractions containing RRF-DI were concentrated by Centricon YM-3 (Millipore), and
the concentrate was further purified by a Superdex 75 pg column (Amersham Biosciences) equilibrated with buffer C containing 150 mM sodium acetate. RRF-DI was purified to homogeneity
(>98%) as judged by SDS-PAGE.
Crystallographic data statistics for V. parahaemolyticus RRF
-amino groups. Acetate buffer (10 mM sodium acetate, pH 4.5) was used as the running buffer
at a flow rate of 10 µl/min. To immobilize RRF-DI onto flow cell 1 of
the CM5 sensor chips, RRF-DI (0.1 mg/ml) was injected as a 24-min
pulse. The level of immobilized RRF-DI was ~5000 resonance units. To
prepare a blank cell, flow cell 2 was also activated but only running
buffer was injected. Unreacted N-hydroxysuccinimide groups
on both cells were blocked with ethanolamine hydrochloride.
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ABSTRACT
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DISCUSSION
REFERENCES
35 element and
Shine-Dalgarno sequence were also included upstream of the open reading
frame in this fragment. The open reading frame consisted of 555 bp and
coded a protein that consists of 185 amino acids. The calculated
molecular weight was 20570.45. Homology between the V. parahaemolyticus and E. coli RRFs was 70.1% at the
amino acid level. The DNA sequence of V. parahaemolyticus
frr has been deposited in the DDBJ/GenBankTM/EMBL
(accession number AB064319).
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Fig. 1.
Comparison of amino acid sequences
from prokaryotic RRFs. The amino acid sequences of RRFs were
aligned with the Gene Web II program (18). The numbering at the
top is according to the sequence from V. parahaemolyticus RRF. Amino acid sequences are shown in single
letter codes. Residues that are conserved in all prokaryotic RRFs are
shown in red. The secondary structure of RRF is depicted
below the alignment. V. pa, Vibrio
parahaemolyticus; V. ch, Vibrio cholerae; E. co,
Escherichia coli; P. ae, Pseudomonas aeruginosa;
C. je, Campylobacter jejuni; H. py, Helicobacter
pylori; M. tu, Mycobacterium tuberculosis; M. le,
Mycobacterium leprae; S. au, Staphylococcus
aureus; A. ae, Aquifex aeolicus; T. th, Thermus
thermophilus; T. ma, Thermotoga maritima.
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Fig. 2.
Ribosome recycling activity of RRFs. The
decomposition of the post-termination complex by RRF from different
sources was examined using E. coli polysome and EF-G.
Closed circles, E. coli; open circles,
V. parahaemolyticus; closed squares, T. thermophilus; open squares, T. maritima.
-helices and six
-strands. The topology of V. parahaemolyticus RRF is nearly identical to other RRFs.
The dimensions of domain I are ~60 × 25 × 25 Å. This
domain is composed of helices
-1,
-4, and
-5 forming a
three-helix bundle, which consists of residues 1-24, 107-144, and
150-182. The three helices are tightly packed against each other
through hydrophobic interactions. Domain II has a globular structure
with a four-stranded anti-parallel
-sheet, a two-stranded
anti-parallel
-sheet, and two short
-helices. These secondary
structure elements form a hydrophobic core. The dimensions of domain II
are about 26 × 22 × 20 Å.
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Fig. 3.
Tertiary structure of V. parahaemolyticus RRF. Stereo view of the structure of
the V. parahaemolyticus RRF with MolScript (44). Elements of
secondary structure are colored as follows: -helix,
green;
-strand, red.
-helices or
-sheets, twelve other hydrogen bonds were
identified in the structure. Eight interhelix hydrogen bonds contribute
to the packing of the
-helix bundle of domain I. Four other hydrogen
bonds stabilize domain-domain interactions. The O-
1 of Thr-29,
located at the end of domain I, forms a hydrogen bond to the O-
2 of
Glu-181, located in the helix 5 of domain I. A hydrogen bond is also
present between the main chain N of Ala-32 in the hinge region and the
main chain O of Ala-62 in the loop region of domain II. The guanidinium
moiety of Arg-110 forms hydrogen bonds to the main chain O of
Glu-184 and O of Leu-182. The significance of these interactions is
shown by the fact that the R110H mutant of E. coli RRF is
nonfunctional (15). The interdomain salt bridge between the N-
atom
of Arg-28, located in the hinge region, and C-
of Asp-86, located in
the loop of domain II, also contributes to the L-shaped structure.
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Fig. 4.
Comparison of V. parahaemolyticus RRF with other RRFs. A,
comparison of RRF structures. RRF Structures are superimposed for best
fit over backbone C atoms of domain I. For A. aeolicus, T. thermophilus, and T. maritima
RRFs, a structural best-fit superposition to V. parahaemolyticus RRF with the C
atoms of domain I (residues
1-30, 104-185) gives values of r.m.s.d. of 1.379, 0.789, and 0.975 Å, respectively. The corresponding best-fit superposition for domain
II (residues 31-103) yields values of r.m.s.d. of 1.360, 1.112, and
1.053 Å, respectively. B, distribution of interdomain
angles for the four crystal structures of RRFs and for the ensembles of
the 15 NMR-derived structures of RRF.
,
, and X defined by Yoshida et al. (10). In the crystal structures, the zenith angles
(
s) are almost 90 °, which corresponds to precise
L-shaped structure. The 110 ° angle for E. coli RRF is an exception, and it has an open L-shaped
structure. Even in the ensemble of solution structures of A. aeolicus RRF,
s are ~90 °. Thus a detergent molecule
(decyl-
-D-maltopyranoside), which was reported to attach
on E. coli RRF, may be responsible for the altered angle in
the crystal structure of E. coli RRF. We concluded that RRFs
from both mesophilic and thermophilic bacteria have a strict
L-shaped structure. We have reported for A. aeolicus RRF that the
angle fluctuates from
30 ° to
30 ° in the ensemble of solution structures. Interestingly, azimuth
angles (
s) among the four crystal structures are spread over a wide
range of ~50 °, but these values fall within the range of solution
structures. Thus we postulated an intramolecular movement within the
RRF molecule where only the
angle fluctuates, while the
angle
is maintained at 90 °. This movement was confirmed by molecular
dynamics calculations.2
Therefore these crystal structures are regarded as snapshots with
variable
angle in solution.
-helix with a double
minimum at 208 and 222 nm. The ellipticity at 222 nm of RRF-DI
represents a higher
-helical content than wild-type RRF. The
deconvolution analysis on this CD curve with the DICROPROT program (32)
indicated an
-helix content of 91%. This indicates that a major
part of RRF-DI consists of
-helix and that the inserted tripeptide
fits nicely into the loop connecting the two
-helical fragments to
form a three-stranded
-helical bundle, which is likely to be quite
similar to that of domain I in wild-type RRF.
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Fig. 5.
Circular dichroism spectra. Far-UV CD
spectra of 5 µM RRF-DI (solid line) and
wild-type RRF (dashed line) in 20 mM sodium
acetate (pH 3.6) and 50 mM sodium chloride. The ellipticity
at 222 nm of RRF-DI represents higher -helical content in RRF-DI
than in wild-type RRF.
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Fig. 6.
Polysome breakdown assay of RRF-DI. The
decomposition of the post-termination complex by RRF-DI was examined
using E. coli polysome and EF-G. Inhibition of wild-type RRF
by RRF-DI was also investigated. (1), no RRF-DI and
wild-type RRF; (2), 1 µM wild-type RRF;
(3), 50 µM RRF-DI; (4), 50 µM RRF-DI plus 1 µM wild-type RRF.
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Fig. 7.
Binding of RRF to 70 S ribosome or
each subunit. A, sensorgrams on Biacore of RRF-DI or
RRF binding to ribosome. Various concentrations of 70 S ribosome or its
subunits (blue, 60 nM; green, 200 nM; red, 900 nM) were injected over
surface with immobilized RRF-DI and wild-type RRF. Bindings of RRF-DI
to the 30 S subunit (a), to the 50 S subunit (b),
and to 70 S ribosome (c) were represented. Bindings of
wild-type RRF to the 30 S subunit (d), the 50 S subunit
(e), and 70 S ribosome (f) were also shown.
B, prohibition of wild-type RRF to the 50 S subunit by RRF-DI. Sensorgrams of RRF binding to the 50 S
subunit (60 nM) in the presence of various concentrations
of RRF-DI are shown: 1, no RRF-DI; 2, 0.05 µM; 3, 0.3 µM; 4, 3 µM. C, binding assay
using filtering technique. RRF-DI and wild-type RRF binding to ribosome
was analyzed by Western blotting. RRF-DI and wild-type RRF in the range
of 0.3-4 µM were incubated with presence (+) or absence
( ) of ribosome. D, Scatchard plot of C.
Dissociation constants of RRF-DI and wild-type RRF from the ribosome
and its subunits
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
RRF-ribosome binding model.
A, comparison of surface representation of V. parahaemolyticus RRF (blue) and yeast
tRNAPhe (green) (45). A new concept of mimicry
between RRF and tRNA is shown. Domain I of RRF corresponds to the
acceptor arm of tRNA. B, the models of RRF and ribosome
complex were constructed according to Selmer's proposal (8).
C, new model for the A-site bound RRF according to
A. This arrangement is consistent with biological
findings that domain I of RRF interacts not with the 30 S subunit but
with the 50 S subunit.
In this study, we have shown that wild-type RRF binds to the 50 S subunit and that it does not bind to the 30 S subunit. According to Selmer's model, domain II of RRF should be responsible for the binding to the 50 S subunit. However our experiments using RRF-DI clearly show that the affinity of RRF domain I to the 50 S subunit is equivalent to that of RRF. Therefore we conclude that binding between RRF and the ribosome depends on the interaction between domain I of RRF and the 50 S subunit, and Selmer's model is excluded. In Selmer's model, Arg-132, which is essential for binding to the ribosome in domain I, is located near the concave cavity of 30 S subunit and does not contribute to binding to the 50 S subunit (Fig. 8B).
This finding led us to consider the other aspect of the mimicry model where domain I was superimposed on the acceptor arm of tRNA. Interestingly, although correspondence between domains of RRF and arms of tRNA in our model is in just the reverse relation to that in Selmer's model, these two molecules are nearly identical in both shape and size, as shown in Fig. 8A.
We constructed the ribosome-RRF complex model by replacing the A-site-bound tRNA in the T. thermophilus 70 S ribosome structure (37) with RRF according to this new concept. In this model, the RRF molecule could be placed in a tight fit in the ribosomal A-site, avoiding any spatial overlap with the ribosome. As shown in Fig. 8C, this model, in which domain I binds to the 50 S subunit, is consistent with our biochemical results in this study. Furthermore, all conserved Arg residues, such as Arg-110, Arg-129, Arg-132, and Arg-133 of RRF domain I, which aligns along the long axis of domain I, would be exposed to 23 S rRNA. In RRF, these positively charged residues are important for the interaction with the ribosomal RNA.2
In our superposition, the spatial orientation of ribosome-bound RRF is
such that -strands 4 and 6 would face the ribosomal P-site and the
-3 helix of domain II would face the factor binding site where EF-G
is bound.
It was noted that a hydrophobic patch is located on the tip of domain
II (10). This region consists of conserved aromatic residues, such as
Tyr-44, Tyr-45, and Phe-70. These residues are unusually exposed to
solvent and are surrounded by hydrophilic residues containing conserved
residues such as Gly-46 and Asp-71. The tip region of domain II may
play a crucial role in recognition of the target molecule. Recently,
the significance of the interactions of RRF with EF-G has been reported
based on the fact that Mycobacterium tuberculosis RRF is
inactive in E. coli, but it regains activity upon
co-expression of M. tuberculosis EF-G (38). From the
mutational studies of RRF and EF-G, Ito et al. have proposed
that EF-G motor action is transmitted to RRF (39). As described under
"Results," azimuth angles () between domains can vary in the
range of ~50 °. Such a domain movement or conformational change
may occur upon EF-G binding. Biochemical and structural studies showed
that the flexibility of the relative orientation of domains I and II
may be important for RRF function (9, 10, 12).
It has been proposed as a hypothetical mechanism that RRF may be bound first to the A-site of the ribosome and then translocated by EF-G to the P-site in a manner similar to that of tRNA, leading to the disassembly of the post-termination complex (7). This model has been based on the fact that RRF binds to the A-site (40) and that tRNA is released in the recycling step (41). We examined whether the mechanism is consistent with the new model. Joseph and Noller reported that the anticodon stem loop of tRNA is required in the A-site for translocation by EF-G during the elongation step (42). However in our model, RRF lacks the part corresponding to the anticodon stem loop of tRNA. Therefore RRF is not likely to be translocated from the A-site to the P-site by EF-G. Furthermore it was shown that the release of tRNA from post-termination complex partially takes place with EF-G alone (35). Therefore, we propose that RRF does not go through a translocation from the A-site to the P-site with the help of EF-G. In this respect, RRF is not a perfect functional tRNA mimic. Movement toward the P-site or conformational change of domain II might assist tRNA release from post-termination complex by EF-G, while domain I still keeps the A-site occupied to protect the A-site against the incoming EF-Tu-aminoacyl-tRNA complex during the disassembly reaction. Using the post-termination complex involving short synthetic mRNA with a Shine-Dalgarno sequence, Karimi et al. have shown in another sequence of ribosome recycling that RRF, EF-G, and GTP catalyze the dissociation of the 50 S subunit from the post-termination complex followed by tRNA removal from the 30-S-deacylated tRNA-mRNA complex by IF3 (43). This may be due to the strong Shine-Dalgarno sequence and may be different from the natural long mRNAs discussed in this study (35).
We have pointed out that movement of the angle that maintains the
L-shaped structure is important for RRF action (10). Based
on this view, the physicochemical study to elucidate the difference in
RRF activity between mesophilic and thermophilic bacteria is in
progress. In this paper, we have proposed a new concept for molecular
mimicry by RRF and a new model for RRF action.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Suzuki, N. Igarashi, and N. Sakabe at the Photon Factory in the High Energy Accelerator Research Organization at Tsukuba, Japan for help with data collection. We also thank Dr. S. Kubo and Y. Takada for technical assistance and useful discussion.
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FOOTNOTES |
---|
* This study was partly supported by Grant-in-Aid 2426 for Japan Society for the Promotion of Science (to H. N.).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 Fig. S1 titled "The
sensorgrams on Biacore of RRF-DI and wild-type RRF binding to ribosome."
The atomic coordinates and the structure factors (code 1IS1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB064319.
¶ Member of the Structural Biology Sakabe Project.
To whom correspondence should be addressed. Tel.:
81-6-6879-8220; Fax: 81-6-6879-8224; E-mail:
yujik@protein.osaka-u.ac.jp.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M208098200
2 T. Yoshida, S. Oka, S. Uchiyama, H. Nakano, T. Ohkubo, and Y. Kobayashi, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RRF, ribosome recycling factor; EF-G, elongation factor G; kbp, kilobase pair; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean square deviations; CD, circular dichroism.
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