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Journal of Bacteriology, November 2000, p. 6154-6160, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Inhibitory Effect of Heterologous Ribosome
Recycling Factor on Growth of Escherichia coli
Kenji
Atarashi and
Akira
Kaji*
Department of Microbiology, School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Received 27 January 2000/Accepted 11 July 2000
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ABSTRACT |
Ribosome recycling factor (RRF) of Thermotoga maritima
was expressed in Escherichia coli from the cloned T. maritima RRF gene and purified. Expression of T. maritima RRF inhibited growth of the E. coli host in
a dose-dependent manner, an effect counteracted by the overexpression
of E. coli RRF. T. maritima RRF also inhibited the E. coli RRF reaction in vitro. Genes encoding RRFs from
Streptococcus pneumoniae and Helicobacter
pylori have been cloned, and they also impair growth of E. coli, although the inhibitory effect of these RRFs was less
pronounced than that of T. maritima RRF. The amino acid
sequence at positions 57 to 62, 74 to 78, 118 to 122, 154 to 160, and
172 to 176 in T. maritima RRF differed totally from that of
E. coli RRF. This suggests that these regions are important
for the inhibitory effect of heterologous RRF. We further suggest that
bending and stretching of the RRF molecule at the hinge between two
domains may be critical for RRF activity and therefore responsible for
T. maritima RRF inhibition of the E. coli RRF reaction.
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INTRODUCTION |
Protein synthesis consists of three
steps: initiation, peptide chain elongation, and termination with the
release of the completed peptide chain. The step that follows these
three steps, disassembly of the posttermination complex, is less well
known but is essential for the next round of protein synthesis (for
reviews, see references 15, 18, 20, and 21). Two
factors, EF-G and ribosome recycling factor (RRF; formerly called
ribosome releasing factor [19]), catalyze this step
(19). In the absence of RRF, ribosomes not only remain on
the mRNA but also resume unscheduled translation downstream of the
termination codon (17, 33).
RRF, discovered in 1970 (7) and confirmed independently in
1973 (40), is encoded by the frr gene, which was
mapped to near 4 min, close to the gene encoding EF-Ts in
Escherichia coli (14). The gene encoding RRF is
found among all living organisms so far examined except for
Archaea (see reviews listed above). Approximately 30 frr genes have so far been sequenced, partly in connection
with bacterial genome sequencing projects (for example, see reference
2), and some RRFs have been characterized (22, 29, 32, 44).
In vitro protein synthesis is stimulated four- to eightfold by the
addition of RRF (27, 31, 34). The GTP requirement (8,
23), the fate of ribosomes at the termination complex in the
presence of RRF (8, 23), and the possible role of IF3 in
certain situations (4, 23, 28) have been studied. In
addition to its role in ribosome recycling, RRF maintains translational
fidelity (18). Although the exact mechanism of RRF action
remains elusive, RRF has been proposed to bind to the ribosomal A site
(20) because it reduces translational error and because its
action is inhibited by the antibiotics which interfere with the A site
(10). RRF competes for ribosomal binding with peptide
release factors that are assumed to bind to the A site (5).
RRF has a nearly perfect structural similarity to tRNA (36).
We therefore proposed that RRF is translocated from the A site to the P
site similarly to tRNA during disassembly of the post- termination
complex (36).
This paper describes the cloning, expression, and purification of RRF
from Thermotoga maritima, which has been used for
determination of the crystal structure (36). In addition,
genes coding for RRF of Streptococcus pneumoniae and
Helicobacter pylori were isolated and studied. In contrast
to Pseudomonas aeruginosa frr, which functions in E. coli (29), we found that these other heterologous RRFs
were toxic to E. coli. We suggest that freezing at the hinge region of the T. maritima RRF structure may be partly
responsible for the toxic effects.
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MATERIALS AND METHODS |
Strains and plasmids.
Table 1
shows all strains and plasmids used in this study. Vectors are pUC19
(Apr) (for carrying E. coli frr), pET-11a
(Apr) (for carrying T. maritima frr), pET-24a(+)
(Kmr) (for carrying S. pneumoniae frr or
H. pylori frr), and pHSG299 (Kmr) (for carrying
E. coli frr). Plasmid pHSG299 (41)) was purchased from Takara. The pHSG299 sequence GenBank accession number is SYNHSG299. The plasmids carrying various frrs are pRR2
(pUC19 carrying E. coli frr), pKA1 (pET-11a carrying
T. maritima frr), pKA5 [pET-24a(+) carrying S. pneumoniae frr], pKA6 [pET-24a(+) carrying H. pylori
frr], and pRR3 (pHSG299 carrying E. coli frr).
BL21(DE3)pLysS is a lysogen of lambda phage derivative DE3, which
carries the gene for T7 RNA polymerase under the control
of the
inducible lacUV5 promoter in the chromosome. T7 RNA polymerase
induced
by the addition of IPTG
(isopropyl-
-
D-thiogalactopyranoside)
drove the
expression of various
frrs. BL21(DE3)pLysS contains
plasmid pLysS expressing T7 lysozyme. This enzyme is a natural
inhibitor of T7 RNA polymerase and reduces its ability to transcribe
target genes in noninduced
cells.
Oligonucleotide primers for PCR.
All oligonucleotide primers
for PCR were synthesized using Beckman Oligo 1000M. The sense and
antisense primers for cloning T. maritima frr were
5'-AGG GGA TAC ATA TGG TTA ATC CGT TCA-3'
and 5'-GCA ACG TGC TGT GGG ATC CTC AAA
ATT-3', respectively. The sense and antisense primers for cloning
S. pneumoniae frr were 5'-GGA ATA AGA AAG CAT
ATG GCT AAC GCA-3' and 5'-GAG TTT TTC TGT
GGA TCC TTA GAC TTC-3', respectively. The sense and
antisense primers for cloning H. pylori frr were 5'-AAA AAG GAT GAA AAC ATA TGT TAC AGG-3' and
5'-CCT TAA TAT CGA ATT CTT AGA CCT TTA-3'. These
primers were designed according to their published sequences
(42) and those given in The Institute for Genomic Research
(TIGR) microbial database on the World Wide Web
(http://www.tigr.org/tdb/mdb/mdb.html). The NdeI,
BamHI, and EcoRI sites are underlined, while the
initiation codons are in boldface italics.
DNA amplification, preparation of plasmids, cloning, and sequence
analysis.
Genomic DNAs of T. maritima were prepared as
described previously (12). Mikhail Shchepetov, University of
Pennsylvania, kindly provided the genomic DNAs of S. pneumoniae. A genomic DNA fragment of H. pylori was
provided by TIGR through the American Type Culture Collection. PCR
amplifications of the frr DNAs of T. maritima, S. pneumoniae, and H. pylori were
carried out with Taq DNA polymerase (Stratagene) using the
primers described in the preceding section. The frr DNA
fragments thus obtained were treated with NdeI and
BamHI (T. maritima and S. pneumoniae)
or NdeI and EcoRI (H. pylori).
Each
frr DNA was excised at the introduced restriction
enzyme sites and was ligated into pET-11a (
T. maritima) or
pET-24a
(+) (
S. pneumoniae or
H. pylori) to
obtain pKA1 or pKA5 and pKA6,
respectively. The inserts of
T. maritima and
S. pneumoniae frr DNA in the pET vector
were sequenced by our DNA Sequencing Facility
(Department of Genetics,
University of Pennsylvania) with the
vector-specific oligonucleotide
primers (corresponding to the
T7 promoter and
terminator).
Expression of various heterologous frrs in E. coli.
Plasmid pKA1, pKA5, pKA6, pRR3, pHSG299, or pRR2 was placed
in BL21(DE3)pLysS (host), depending on the experiment. The host harboring the plasmid was grown at 37°C overnight in Luria-Bertani (LB) medium containing the appropriate antibiotic to select against the
emergence of the strain without the plasmid. These antibiotics were
ampicillin (50 µg/ml for pKA1, pRR2, and pUC19), kanamycin (50 µg/ml for pKA5, pKA6, pRR3, and pHSG299), and chloramphenicol (25 µg/ml for pLysS). The cultures were diluted 100-fold with fresh LB
medium supplemented with the same antibiotics and shaken at 37°C to
early mid-log phase (optical density at 600 nm
[OD600] > 0.5).
Cells from a 1-ml culture were collected by centrifugation and
suspended in 50 µl of water-50 µl of 2× loading buffer for
sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and
heated in a boiling water bath. The lysates were applied to
a 15%
acrylamide gel, which was subjected to electrophoresis at
30 mA as
described previously (
35), followed by staining with
Coomassie brilliant blue
R.
Purification and antigenic properties of T. maritima
RRF.
E. coli BL21(DE3)pLysS harboring pKA1 carrying
T. maritima frr was grown in 4 liters of LB medium
supplemented with appropriate antibiotics. Cells were induced with 1 mM
IPTG for 4 h and collected, and the crude extract (136.6 mg) was
obtained as described previously (9). The heat-treated
extract (1.9 mg/ml; total, 16 ml; purity, 66.5%) was applied on a
Sephadex G-100 column (15 g of Sephadex G-100 superfine [Pharmacia];
2.6 by 40 cm). The column had been equilibrated with buffer J (10 mM
Tris-HCl [pH 7.6], 50 mM NH4Cl, 10 mM MgSO4,
0.5 mM dithiothreitol), and RRF was eluted with the same buffer at 4 drops/min at 4°C.
Fractions (4.7 ml/tube) were collected. The eluates were concentrated
by Centriprep 10 (Amicon) and examined by SDS-PAGE.
The protein
concentration was measured with the DC protein assay
kit (Bio-Rad).
Western blotting was performed as described previously
(
35).
RRF assay.
For disassembly of the posttermination complex, a
model substrate was prepared from polyribosomes isolated from growing
E. coli in the presence of tetracycline, which inhibits
binding of aminoacyl tRNA to the ribosomal A site. The polysomes have
nascent growing polypeptides, which are removed by puromycin. The
resulting complex consists of ribosomes with deacylated tRNA on the P
site, E site, and an empty A site. Each ribosome in this polysome can be regarded as a model for the posttermination ribosomal complex. The
only difference between this model and the actual posttermination complex is that the real posttermination complex has a termination codon at the A site while the ribosome isolated from the polysome can
have any codon at this site. Since disassembly of the posttermination complex does not depend on a specific codon, this model substrate is
adequate for examining the RRF reaction (9).
Disassembly (release of ribosomes from mRNA) of the model complex was
measured by the conversion of polysomes to monosomes.
The conversion is
detected by monitoring the sedimentation profile
of the ribosomes in
sucrose gradient centrifugation. Polysomes
and purified
E. coli RRF were prepared as described previously
(
9).
EF-G was prepared using the purification method previously
described
(
11,
25). A typical reaction mixture (275 µl) for
the in
vitro assay of RRF contained 10 mM Tris-HCl (pH 7.4), 8.2
mM
MgSO
4, 80 mM NH
4Cl, 6 mM dithiothreitol, 0.16 mM GTP, 0.05
mM puromycin, 0.719
A260 units of
polysome, 19 µg of EF-G, and
5 µg of
E. coli RRF.
Various amounts of
T. maritima RRF were
added.
The reaction mixture was incubated at 30°C for 15 min, and the
conversion of polysomes to monosomes was examined as follows.
The
mixture was placed on top of 5 ml of sucrose gradient (15
to 30%) and
centrifuged for 90 min at 40,000 rpm at 4°C in a Beckman
SW 50 rotor.
The gradient was analyzed by measuring UV absorption
at 254 nm using
the ISCO gradient analyzer. The release of ribosomes
from mRNA was
monitored by measuring the increase in the amount
of 70S ribosomes and
the decrease of polyribosomes. Depending
on the preparation of
polysomes, some 70S monosomes were already
present in the preparation
before the RRF reaction. The amount
of preexisting monosomes was
subtracted from the amount of monosomes
present after the RRF reaction
to calculate the conversion of
polyribosomes to monosomes due to the
RRF reaction. Details of
the assay procedure have been described
previously (
9).
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RESULTS |
Purification of T. maritima RRF and the antigenic
properties of purified T. maritima RRF.
E.
coli BL21(DE3)pLysS cells harboring pKA1 carrying T. maritima frr were treated as described in Materials and Methods,
and crude T. maritima RRF with a purity of 23.5% was
obtained. For further purification, the crude T. maritima
RRF was incubated at 75°C for 30 min to remove the bulk of the
proteins from the host cells as shown in Fig.
1A. By this procedure, extracts (30.5 mg
of protein of which 66.5% was RRF) were obtained. A similar treatment
was equally effective for the purification of other T. maritima proteins expressed in E. coli (30, 39,
43). The heat-treated extract was then applied to a Sephadex
G-100 column. The representative fractions were examined for purity as
shown in Fig. 1B. The purified T. maritima RRF (5.5 mg/ml
and 92.0% purity) was thus obtained with 25.0% recovery. Total
recovery over the entire purification procedure was 15.8%.
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FIG. 1.
Purification of T. maritima RRF. (A) A
bacterial crude extract was heated at 75°C for 30 min, and the crude
extracts obtained before (pre) and after (post) the heat treatment were
analyzed by SDS-15% PAGE followed by staining with Coomassie
brilliant blue R. (B) The heated extracts were loaded on a Sephadex
G-100 superfine (Pharmacia Biotech) column, and representative
fractions were examined by SDS-PAGE as described for panel A. MW,
molecular weight markers (GIBCO).
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The cross-reactivity of purified
T. maritima RRF with an
anti-
E. coli RRF polyclonal antibody was examined as shown
in Fig.
2. Although
T. maritima RRF cross-reacted with the anti-
E. coli RRF
antibody, the reactivity was at least 300-fold less than that
of
E. coli RRF.
T. maritima RRF is therefore only
remotely related
to
E. coli RRF immunologically. It is noted
that
T. maritima RRF
migrated more slowly than
E. coli RRF (see also Fig.
6A), indicating
that the band observed in
lane 1 represents
T. maritima RRF and
moved slower than that
of
E. coli RRF.
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FIG. 2.
Antigenic cross-reactivity of T. maritima RRF
with anti-E. coli RRF antibody. Purified RRFs were analyzed
by SDS-15% PAGE, and the Western blotting was performed with
anti-E. coli RRF antibody. Lane 1, T. maritima
RRF, 3,000 ng; lane 2, E. coli RRF, 30 ng; lane 3, E. coli RRF, 10 ng; lane 4, prestained protein marker, broad range
(BioLabs).
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Inhibitory effect of T. maritima, S. pneumoniae, and H. pylori RRF on the growth of
E. coli.
We examined the effect of T. maritima
frr expression on the growth of E. coli (Fig.
3). The pET-11a vector carried T. maritima frr with the ampicillin resistance gene
(Apr). The expression of T. maritima frr was
induced by the addition of IPTG. Induction of T. maritima
frr inhibited the growth of E. coli (Fig. 3A and D).
The maximum killing effect was such that 90% of the viable counts were
lost; the data represent typical results that were reproduced several
times. The inhibitory effect depended on the concentration of the
inducer, suggesting a dose-dependent relationship in the inhibition by
T. maritima frr. The inhibitory effect appears to affect the
viability of the host more than the increase in OD. Similarly,
inactivation of temperature-sensitive (ts) RRF had a greater pronounced
effect on viability than on the ODs of the cell cultures
(17).
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FIG. 3.
T. maritima RRF inhibits the
growth of E. coli. E. coli BL21(DE3)pLysS cells
(host), harboring various plasmids as indicated below, were grown at
37°C overnight in LB medium supplemented with appropriate
antibiotics. The overnight culture was diluted 100-fold and grown at
37°C. At various times after IPTG addition, the OD600 of
the culture (A to C) and viable counts (D to F) were measured. (A and
D) Presence of T. maritima frr inhibits E. coli.
Solid triangles, noninduced host harboring pKA1 and pHSG299 (empty
vector, control for pRR3); open triangles, 1 mM IPTG-induced host
harboring pKA1 and pHSG299; inverted triangles, same as open triangles
except 0.1 mM IPTG was used for induction of T. maritima
RRF. (B and E) Presence of extrachromosomal E. coli frr
neutralizes toxic effect of T. maritima frr. Open circles,
IPTG (1 mM)-induced host harboring pKA1 and pRR3 (pHSG299 carrying
E. coli frr and the Kmr gene); inverted
triangles, same as open circles except 0.1 mM IPTG was used; solid
circles, noninduced host harboring pKA1 and pRR3. (C and F) Empty
vectors and extrachromosomal E. coli frr have no effect on
E. coli. Squares, host harboring pET-11a (empty vector,
control for pKA1) and pRR3 in 1 mM IPTG; diamond, 1 mM IPTG was added
to host harboring pET-11a and pHSG299 (control for pRR3).
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The presence of multicopy
E. coli frr reversed the
inhibitory effect of
T. maritima frr (Fig.
3B and E). These
cells were
identical to those used in Fig.
3A but contained plasmid
pRR3,
which carried
E. coli frr and kanamycin resistance.
These figures
show that a functional extrachromosomal
E. coli
frr overcame in
large part the toxic effect of
T. maritima RRF. The empty vectors
used in these experiments, as well
as the presence of pRR3, had
no deleterious effect on the host cells,
as shown in Fig.
3C and
F.
Figures
4 and
5 show
similar but less-pronounced effects by
S. pneumoniae and
H. pylori frr, respectively. In both cases,
the addition of
1 mM IPTG exerted a deleterious effect, whereas,
in the absence of
IPTG, bacterial growth was identical to that
for the controls (Fig.
4A
and C and 5A and C). The effect of
H. pylori frr,
however, was so mild that it did not reduce the viable
count but only
retarded the increase (Fig.
5A and C). These toxic
effects were
significantly reduced when plasmids carrying
E. coli frr
were present simultaneously (Fig.
4B and D and 5B and D).
Since the
expression of
E. coli frr carried by pRR2 did not depend
on
adding inducer (Fig.
6A, lane 3)
(
13,
38), the constant
presence of excess RRF is not toxic.
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FIG. 4.
Inhibitory effect of S. pneumoniae
frr on growth of E. coli. (A and C) S. pneumoniae
frr inhibits the growth of E. coli. Open triangles, 1 mM IPTG-induced E. coli BL21(DE3)pLysS (host) harboring
pKA5 [pET-24a(+) carrying the kanamycin resistance gene and S. pneumoniae frr) and pUC19 (empty vector, control for pRR2); solid
triangles, same except no IPTG was added. (B and D) Extrachromosomal
E. coli frr reduces the toxic effect of S. pneumoniae
frr. Open circles, IPTG-induced host harboring pKA5 and pRR2
(pUC19 carrying E. coli frr and the ampicillin resistance
gene); solid circles, same except no IPTG was added.
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FIG. 5.
Inhibitory effect of H. pylori frr on
E. coli growth. (A and C) H. pylori frr inhibits
E. coli growth. Open triangles, 1 mM IPTG-induced E. coli BL21(DE3)pLysS (host) harboring pKA6 [carries H. pylori frr in pET-24a(+)] and pUC19 (empty vector, control for
pRR2); solid triangles, same except no IPTG was added. (B and D)
Presence of extrachromosomal E. coli frr reduces the toxic
effect of H. pylori frr. Open circles, IPTG-induced host
harboring pKA6 and pRR2 (pUC19 carrying E. coli frr); solid
circles, same except no IPTG was added.
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FIG. 6.
Expression of RRF of various bacterial origins in
E. coli. Whole-cell lysates of E. coli strain
BL21(DE3)pLysS (host) expressing RRF of various origins were
analyzed by SDS-PAGE. Molecular weight markers (GIBCO) (A, lane 6, and
B, lane 5) and 5 µg of pure E. coli RRF (A and B, lanes 1 are shown for comparison in each of the figures. (A) Expression of
T. maritima and E. coli RRF. Lane 2, host
harboring pKA1 (carries T. maritima frr in pET-11a) and pRR3
(carries E. coli frr in pHSG299) were induced with 1 mM
IPTG; lane 3, same as lane 2 except no IPTG was added; lane 4, host
harboring pKA1 and pHSG299 (empty vector, control for pRR3) was induced
with 1 mM IPTG; lane 5, same as lane 4 except that no IPTG was added.
(B) Expression of H. pylori and E. coli RRF. Lane
2, host harboring pKA6 [carries H. pylori frr in
pET-24a(+)] and pRR2 were induced with 1 mM IPTG; lane 3, host
harboring pKA6 and pUC19 (empty vector, control for pRR2) were induced
with 1 mM IPTG; lane 4, same as lane 3 except that no IPTG was added.
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Evidence for the expression of heterologous RRF in E. coli.
The preceding results can be interpreted by assuming that
each of the plasmids carrying non-E. coli frr expresses the
corresponding RRF. Indeed, plasmids pKA1 (pET-11a carrying
T. maritima frr) and pRR3 (pHSG299 carrying
E. coli frr) expressed the respective RRFs (Fig. 6A, lane
2). The relative amounts of these RRFs were in the ratio of 10 (E. coli)/8 (T. maritima) (average of
the densities of the bands on three different gels). Because E. coli RRF migrated slightly faster than that of T. maritima, one can detect the presence of both. That the
slower-moving band corresponds to T. maritima RRF is clear
from lanes 3 to 5. We conclude from this experiment that expression of
the E. coli frr gene neutralizes the deleterious effect of
T. maritima RRF.
In a similar manner, under the conditions where the plasmid carrying
E. coli frr reduced the deleterious effect of S.
pneumoniae frr, the expression of both RRFs indeed took
place. The relative
amounts of these RRFs in this cell were 7 (
S. pneumoniae) to 10
(
E. coli) (data
not
shown).
Although
H. pylori RRF shows the weakest inhibition of
E. coli growth, this is not due to a weak
expression of
H. pylori RRF,
as shown in lane 3 in Fig.
6B.
We conclude that the relative amount
of
H. pylori RRF
expressed was similar to that of
T. maritima RRF (compare
Fig.
6B and
A).
Inhibition of the E. coli RRF reaction by T. maritima RRF.
We examined the effect of T. maritima RRF on the in vitro RRF assay using naturally occurring
polysomes (9). In this system, polyribosomes isolated from
growing E. coli cells were treated with puromycin to remove
the nascent peptide. We regard the resulting complex of ribosome, tRNA,
and mRNA as a model substrate for the posttermination ribosomal
complex. Disassembly of this model posttermination complex converts the
polysomes into monosomes (8).
Table
2 shows that the strongest
inhibition (65%) of
E. coli RRF by
T. maritima
RRF was observed when the largest amount
of
T. maritima RRF
(10-fold larger than the amount of
E. coli RRF) was added.
Equal amounts of
T. maritima RRF and
E. coli RRF
in the reaction mixture yielded only a slight (7%) inhibition.
This
result suggests that the affinity of
T. maritima RRF
for
the
E. coli polysomes must be less than 10% that of
E. coli RRF.
T. maritima RRF did not disassemble
the posttermination complex
of
E. coli (Table
2).
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DISCUSSION |
It is known that P. aeruginosa RRF (29) and
the L-lactate dehydrogenase gene from T. maritima (30) function in E. coli. We
therefore expected that heterologous RRFs would function in E. coli because their sequences are very similar to that of E. coli RRF (Fig. 7). Contrary to this
expectation, expression of the genes coding for these RRFs was
deleterious to E. coli. We suggest that this toxic
effect is due to an inhibitory action of a heterologous RRF on the
reaction catalyzed by E. coli RRF for the following
reasons.
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FIG. 7.
Comparison of amino acid sequences of RRFs of various
origins. Amino acid sequences of RRFs were aligned using the Clustal V
program (6). Ecol, E. coli (13) (GenBank accession no.
J05113); Paer, P. aeruginosa (29) (DDBJ accession no.
AB010087); Tmar, T. maritima (GenBank accession no.
AAD36470); Spne, S. pneumoniae (TIGR microbial database;
http://www.tigr.org/tdb/mdb/mdb.html). Hpyl, H. pylori (42) (GenBank accession no. P56398). Identity (*) and
similarity (·) based on the Dayhoff PAM-250 matrix, are indicated
(1). Shaded residues, identity with the sequence of E. coli RRF.
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First, the toxic effects of the heterologous frr depend on
the extent of induction of these heterologous RRFs by IPTG. Second, analysis of the E. coli extract harboring the heterologous
frr showed the presence of the corresponding
heterologous RRF. Third, pure heterologous RRF represented by
T. maritima RRF inhibited the in vitro E. coli RRF reaction. Fourth, the heterologous RRF, represented by
T. maritima RRF, was close enough to E. coli RRF immunologically to be cross-reactive. Although cross-reactivity does
not necessarily indicate a similar structure, it suggests that the
heterologous RRF can compete with E. coli RRF because of the
structural similarities of these two proteins. Fifth, and most
importantly, the simultaneous expression of an excess amount of
E. coli RRF overcame the toxic effect of the heterologous
RRF. In addition, T. maritima frr did not complement LJ15
(17), an E. coli mutant carrying ts RRF at
47°C (the nonpermissive temperature of this ts RRF) (data not shown).
We previously reported that spinach RRF exerted a deleterious effect on
a mutant E. coli carrying a ts RRF (32). The
finding reported here represents the first observed inhibitory effect
of a heterologous RRF on wild-type E. coli.
Figure 7 shows the amino acid sequences of RRF from E. coli (13), P. aeruginosa (29),
T. maritima (36; see Addendum in Proof),
S. pneumoniae, and H. pylori
(42). In the five sequence segments (a to e) there are many
amino acids of E. coli RRF identical to those of P. aeruginosa RRF but the RRFs of the other species showed only a few
identical amino acids. This suggests that these segments may be
responsible for the inhibitory effect of the heterologous RRFs.
We have recently resolved the crystal structure of T. maritima RRF prepared as described in this paper; it consists of
two domains, domain I being a long three-helix bundle and domain
II being a three-layer -
- sandwich (36). Recent
work on E. coli RRF (26) confirmed this
structure, which essentially agrees with the computer-predicted
secondary structure (3) of RRF (16). These two
domains are connected through the hinge region. Out of the five
segments marked in Fig. 7, segments a and b are in domain II while the
remaining three are in domain I.
A heterologous RRF may act on E. coli RRF directly and
inactivate it by forming an inactive complex. Our preliminary nuclear magnetic resonance studies on RRF (24), however, indicate
that RRF tends to stay as a monomer. In addition, it takes at least a
10-fold-higher molar concentration of heterologous RRF for a 50%
inhibition of E. coli RRF (Table 2). Since RRF is a nearly perfect mimic of tRNA, we postulated that it behaves like tRNA on the
ribosome (36). Our mutation data (17) support
this hypothesis. It is therefore more likely that the heterologous RRF
competes with E. coli RRF for the ribosomal A site (the site at which aminoacyl tRNA binds to the ribosome).
Why then does the A site-bound T. maritima RRF not work for
E. coli? We may speculate that RRF must bend at the hinge
region during its action. It is possible that T. maritima
RRF can bend at 80°C but not at 37°C because molecular flexibility
increases with temperature. This makes it difficult for T. maritima RRF bound at the A site to function at 37°C. The
following observations lead to this speculation. First, crystallization
of E. coli RRF depends on
decyl--D-maltopyranoside, which fits into the pocket of
the hinge region of RRF (26), probably fixing the
molecule in one form. Second, no such agent is required for
crystallization of T. maritima RRF at room temperature
(37), probably because this RRF keeps itself in one form due
to its nonflexibility at room temperature. This is conceivable because
T. maritima RRF is probably designed to be bendable at the
hinge region at 80°C but not at 37°C. Third, the hinge region
represents the high-mobility region of this molecule (estimated
from the crystal structure) despite the fact that it is not near
the N or C terminal. It should be noted that less pronounced
inhibitory effects by S. pneumoniae and H. pylori
RRFs probably do not involve this mechanism because their RRFs are
designed to function at 37°C. For inhibition by these RRFs and
T. maritima RRF, the a-e regions may also play important roles.
| |
ACKNOWLEDGMENTS |
We thank Karl O. Stetter and Robert Huber of the University of
Regensburg, Regensburg, Germany, for providing DNA of T. maritima, James Kocsis of Jefferson Medical College of critically
reading the manuscript and for linguistic help, and Yun-Wen Shaw for
clerical assistance.
| |
ADDENDUM IN PROOF |
The complete sequence of the Thermotoga maritima DNA
has been published (K. E. Nelson et al., Nature
399:323-329, 1999).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Room 319B,
Johnson Pavilion, Department of Microbiology, School of Medicine,
University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA 19104. Phone: (215) 898-8828. Fax: (215) 573-2221. E-mail:
kaji{at}mail.med.upenn.edu.
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Abstract
Introduction
