From the a Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan, the f Department of Parasitology, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-0071, Japan, the h Laboratory of Molecular Life Science (Schering-Plough), Faculty of Biotechnology and Bioscience, Tokyo Institute of Technology, Nagatsuta, Yokohama 227-8501, Japan, and the i Institute of Molecular and Structural Biology, Aarhus University, Gustav Wiedsvej 10C, DK-8000 Aarhus C, Denmark
Received for publication, December 11, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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We have found the gene for a translation
elongation factor Tu (EF-Tu) homologue in the genome of the nematode
Caenorhabditis elegans. Because the corresponding protein
was detected immunologically in a nematode mitochondrial (mt) extract,
it could be regarded as a nematode mt EF-Tu. The protein possesses an
extension of about 57 amino acids (we call this domain 3') at the C
terminus, which is not found in any other known EF-Tu. Because most
nematode mt tRNAs lack a T stem, domain 3' may be related to this
feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for
binding to T stem-lacking tRNAs. This finding may constitute a novel
example of the co-evolution of a structurally simplified RNA and the
cognate RNA-binding protein, the latter having apparently acquired an
additional domain to compensate for the lack of a binding site(s) on
the RNA.
One of the unusual features of the animal mitochondrial
(mt)1 translation system is
the use of tRNA with highly divergent structures (1). Such nonstandard
structural forms have been predicted from gene sequences encoded in mt
DNA (2), and some have been confirmed at the RNA level (3, 4).
Mitochondrial tRNA structural divergence is thought to reflect relaxed
constraints operating on translation systems that produce very few
proteins encoded in mt DNA (5). However, almost all of the
nuclear-encoded protein factors that are anticipated to interact with
such divergent tRNAs remain to be identified.
In the elongation cycle of prokaryotic translation, one of the most
crucial steps is the formation of an active ternary complex among
elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP
followed by transfer of the aa-tRNA to the ribosomal A site (6, 7).
Recent crystallographic analysis (8, 9) as well as a variety of
biochemical data (10) show that bacterial EF-Tu binds mainly to two
regions within tRNA: the terminal region of the acceptor stem and one
side of the T stem helix, the length of which strongly influences
ternary complex formation (11). Similar tRNA binding mechanisms have
been proposed for elongation factor 1 Preparation of cDNAs for Various EF-Tus--
An internal
fragment of A. suum EF-Tu homologue cDNA was obtained
from poly(A)+ RNA prepared from A. suum adult
body wall muscle by reverse transcription with random hexamers as
primers and avian myeloblastosis virus reverse transcriptase (Roche
Molecular Biochemicals). The DNA fragments obtained were further
amplified by PCR using the primers 5'Tu and 3'Tu (Table
I), which correspond to the putative
conserved peptide sequences IGHVDH (amino acid residues 18-23
of Escherichia coli EF-Tu in Fig. 1) and MITGAA (amino acid
residues 93-98). The ~250-base pair-long product was purified by
agarose or nondenaturing polyacrylamide gel electrophoresis,
phospholyated by T4 polynucleotide kinase (Toyobo) and ATP, and then
inserted into the SmaI site of pUC19 (21). The chimeric
plasmid was sequenced with a T7 Sequenase version 2.0/7-deaza-dGTP DNA
sequencing kit (Amersham Pharmacia Biotech) using
[
The C. elegans cDNA clones cm5c1 and cm20d1 (24) (the
respective accession numbers for the partial sequences of these clones are Z14779 and M89378) were kindly provided by J. E. Sulston and
A. Coulson (Sanger Centre, Cambridge, UK), and L. Fulton
(Washington University School of Medicine), respectively. The Construction of Expression Vectors of EF-Tu Variants--
For
expression of recombinant EF-Tus in E. coli cells, a portion
of the C. elegans cDNA clone cm5c1 encoding the amino
acid sequence between 39G and 496P (the N terminus was chosen by
referring to the prokaryotic EF-Tu sequences; see Fig. 1) was first
amplified by PCR using the primers 5'CeTu and 3'CeTuA (Table I) and
then inserted between the NdeI and BamHI sites of
pBluescript II KS+ (Stratagene). The NdeI-BamHI
fragment of the cloned insert was subcloned between the NdeI
and BamHI sites of pET-15b (Novagen).
The Thermus thermophilus EF-Tu gene cloned in pKK223-3 (26)
and bovine mt EF-Tu cDNA cloned in pET-24c(+) (27) were kindly provided by T. Yokogawa (Gifu University, Gifu, Japan) and
L. L. Spremulli (University of North Carolina), respectively.
These genes were amplified by PCR using the primers 5'TthTu and 3'TthTu (Table I) for T. thermophilus EF-Tu and 5'BmTu and 3'BmTu
for bovine mt EF-Tu. Coding sequences for the hybrids of these two proteins (see Fig. 4) were prepared by PCR ligation using two-step PCR.
For the first PCR, the bovine mt/T. thermophilus EF-Tu gene was amplified between 5'BmTu/5'TthTu and either 3'BmCe4, 3'BmCe3, 3'TthCe3', 3'TthCe3, or 3'TthCe2, and also C. elegans mt
EF-Tu cDNA was amplified between either 5'BmCe4, 5'BmCe3,
5'TthCe3', 5'TthCe3, or 5'TthCe2 and 3'CeTu. For the second PCR, the
first PCR products were ligated by PCR using 5'BmTu/5'TthTu and 3'CeTu. All of these primers are given in Table I. All of the coding sequences
of EF-Tu variants cloned into the NdeI-BamHI site
of pET-15b were confirmed by sequencing analysis with an ABI PRISM 310 genetic analyzer.
Recombinant Proteins--
The strain BL21(DE3) or its pLysS
derivative (28) was transformed by expression vectors containing the
EF-Tu variant genes. These transformants allowed the production of
His-tagged proteins, which were then purified by His-Bind resin
(Novagen) or nickel-nitrilotriacetic acid agarose (Qiagen) column
chromatography. The recombinant bacteria were grown at 37 °C in
Terrific broth medium (29) with 100 µg/ml ampicillin (and 23 µg/ml
chloramphenicol when the host cell was BL21(DE3)pLysS) until the
absorbance at 600 nm reached around 0.5. Isopropyl- Preparation of Anti-EF-Tu Homologue Antiserum--
The
recombinant C. elegans protein (39G-496P) with the His tag
sequence obtained above (150 µg/mouse on day 1 as a 50:50 (v/v) mixture with Freund's complete adjuvant (Difco), and 35 and 30 µg on
days 14 and 29, respectively, as a 50:50 (v/v) mixture with Freund's
incomplete adjuvant (Difco) for the boosters) was injected into four
BALB/c mice (female, 5 weeks old) to produce its polyclonal antibodies.
The serum obtained from the immunized mice on day 35 was purified by a
recombinant protein-bound nitrocellulose membrane (BA85; Schleicher & Schüll) according to Roth et al. (30).
Western Blotting--
Cytoplasmic soluble (as a post-mt
supernatant) and mt fractions were prepared from C. elegans
whole bodies as described (31). The protein concentration was estimated
according to Lowry et al. (32) using bovine serum albumin as
a standard. The activity of succinate dehydrogenase as a mt marker
enzyme was 64.5 nmol/min/mg protein in the mt fraction but was not
detectable in the cytoplasmic fraction. The proteins and size markers
(Bio-Rad prestained markers) charged in different lanes were separated
by a 12.5% SDS-polyacrylamide gel electrophoresis, and the resultant
protein fractions were electroblotted onto a nitrocellulose membrane
(BA85). The membrane was blocked with 3% (w/v) gelatin in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% (v/v)
Tween 20) followed by incubation with the affinity-purified antiserum
of the C. elegans EF-Tu for 6 h at room temperature.
Immunodetection using the conjugate between goat anti-mouse IgG(H+L)
antibody and alkaline phosphatase (Bio-Rad) was performed according to
the established protocol (33).
Preparation of Aminoacyl-tRNA--
A. suum mt
tRNAMet was purified as described (20). Partially purified
E. coli tRNAmMet was provided by N. Hayashi (The University of Tokyo) and further purified by a Q Sepharose
(Amersham Pharmacia Biotech) column chromatography or native
polyacrylamide gel electrophoresis. Aminoacylation was carried out
using [35S]Met (200 GBq/mmol) under the same buffer
conditions as described elsewhere (for A. suum mt
tRNAMet, Ohtsuki et al. (20); and for E. coli tRNAmMet, Takemoto et al.
(34)). The charged tRNA was extracted by phenol saturated with 50 mM NaOAc (pH 4.5), precipitated by ethanol (two times), and
dissolved with 20 mM KOAc (pH 4.5) before use.
Deacylation-Protection Assay--
E. coli
EF-Tu was purified according to the literature (35). The
deacylation-protection assay was performed basically according to
Pingoud et al. (36). The reaction mixture (75 mM
Tris-HCl (pH 7.4), 75 mM NH4Cl, 15 mM MgCl2, 7.5 mM dithiothreitol,
0.1 mM GTP, 60 µg/ml bovine serum albumin, 2.25 mM phosphoenolpyruvate, 2.3 units/ml pyruvate kinase,
EF-Tu, and [35S]Met-tRNA) was preincubated without
[35S]Met-tRNA at 30 °C for 5 min, and then
[35S]Met-tRNA was added. Deacylation reaction was
performed at 30 °C.
The C. elegans EF-Tu Homologue Has a Long C-terminal
Extension--
No EF-Tu gene has been identified in the nematode mt
DNAs characterized so far (16, 18), which suggests that it is
nuclear-encoded. To clone the nematode mt EF-Tu, we first amplified the
cDNA from a parasitic nematode, A. suum, by reverse
transcriptase-mediated PCR using primers designed from peptide
sequences that are conserved among bacterial and yeast mt EF-Tus and
then obtained a partial cDNA
fragment.2 The peptide
sequence predicted from the cloned PCR product was significantly
homologous (more than 80%) with the sequences of prokaryotic and
eukaryotic mt and plastid EF-Tus so far reported. A data base search
revealed that the peptide sequences predicted from several partial
cDNA sequences of the free-living nematode C. elegans
(24, 37) had 95% identity with that of our clone. We could thus
determine the complete sequences of the two C. elegans cDNA clones, cm5c1 and cm20d1, whose internal sequences
corresponding to the open reading frame of the EF-Tu were identical to
each other. Although these clones lack the 5' trans-spliced leader (SL)
sequence that occurs at the 5' end of most nematode nuclear-encoded mRNAs, a putative SL1 sequence (38) was found just upstream of the
5' end sequence of the clone cm5c1 by 5'-RACE (25) using a primer
designed from a defined sequence in the 5'-terminal region of the
cDNA clones. It thus became clear that the 5' end sequence of the
clone cm5c1 contained the full open reading frame with the initiation
codon. The C. elegans gene was mapped on chromosome III as
part of the C. elegans genome project (37).
The longest coding region of the cDNA corresponds to a protein
comprised of 496 amino acid residues (Fig.
1), including a region of about 40 residues characteristic of mt transit peptides (39) at the N terminus.
In addition, it contains an extraordinary 57-amino acid-long extension
at the C terminus (domain 3') not found in any other known EF-Tu
sequence (Fig. 1). The molecular mass of a possible mature form of the
protein was estimated to be about 5.1 × 104 (40).
Except for the C-terminal extension, the predicted sequence has all of
the characteristic features common to the mt EF-Tus reported so far
(Fig. 1) (41).
Cellular Localization of the EF-Tu Homologue--
To determine the
cellular localization of the nematode EF-Tu homologue, we prepared both
cytoplasmic soluble and mt fractions from C. elegans cells
and analyzed them immunologically. A protein that could react with the
affinity-purified antiserum obtained from mice immunized with a
presumed recombinant mature protein (see "Experimental Procedures")
was found in the mt extract from C. elegans (Fig.
2). The estimated size (51 kDa)
corresponded well with that predicted for the mature protein without
the mt transit peptide. No such protein reactive with the antiserum was detected in the cytoplasmic fraction from C. elegans (Fig.
2). These results suggest that the cDNA-encoded protein is actually localized in the nematode mt fraction as the mature form.
The C. elegans EF-Tu Homologue Specifically Binds to Nematode mt
tRNA Lacking the T Stem--
To examine the biological function of the
nematode EF-Tu homologue in more detail, we checked its binding
activity toward aa-tRNAs from E. coli and nematode
mitochondria by means of the deacylation-protection assay (36). This
showed that the nematode protein could provide nematode mt
Met-tRNAMet lacking the T stem with considerable protection
against deacylation, whereas E. coli EF-Tu could not (Fig.
3A). On the other hand, E. coli EF-Tu protected E. coli
Met-tRNAmMet from deacylation very well, but
the nematode protein protected it only slightly (Fig. 3B).
Thus, it can be concluded that the nematode EF-Tu homologue is an
actual EF-Tu of the nematode mitochondria.
Domain Exchanges among EF-Tus of Nematode and Mammalian
Mitochondria and Eubacteria--
To further elucidate the function of
each domain of the C. elegans mt EF-Tu in aa-tRNA binding,
we designed and produced a series of chimeric proteins between the
C-terminal domain(s) (domains 3' and/or 3) of the C. elegans
mt EF-Tu and the N-terminal domain(s) (domains 1 and/or 2) of the
eubacterium, T. thermophilus or bovine mt EF-Tus (Fig.
4), and examined their abilities to
protect the nematode mt and eubacterial aa-tRNAs from hydrolysis. As
already reported by Spremulli's group (42), the three domains of EF-Tu and also domains 3 and 3' (Fig. 1) are connected by random coils that
serve as hinges, allowing the domains to move relative to each other.
For this reason, these regions were selected as the junction sites for
constructing the chimeric proteins.
As Fig. 5 shows, although bovine mt EF-Tu
(even the native enzyme) protected the hydrolysis of nematode
Met-tRNAMet only slightly, adding domain 3' of C. elegans mt EF-Tu to the bovine mt counterpart (BmCe3') enhanced
the protection efficiency 2-fold, and further replacement of domain 3 (BmCe3; domains 1 and 2 of the bovine mt EF-Tu were fused with domains
3 and 3' of C. elegans mt EF-Tu) raised the protection
efficiency to the same level as that of nematode EF-Tu (Fig.
5A). On the other hand, when the N-terminal domain(s) of
T. thermophilus EF-Tu was fused with the C-terminal
domain(s) of the C. elegans mt EF-Tu, even after replacement
up to domain 2 by the C. elegans mt EF-Tu domains, the
resultant fusion proteins (ThCe2, ThCe3, and TthCe3') could not bind to
nematode mt tRNA (Fig. 5A). This suggests that not only
domain 3' but also one or more of the other domains are required for
binding to tRNA lacking a T stem.
As for the protection of E. coli
Met-tRNAmMet, bovine mt EF-Tu showed the same
activity as T. thermophilus EF-Tu (Fig. 5B). It should be pointed out that adding domain 3' of the C. elegans mt EF-Tu to either of these EF-Tus (BmCe3' and TthCe3')
did not hinder their protective activity at all, whereas the additional swapping of domain 3 (BmCe3 and TthCe3) completely eliminated such
activity. These results suggest that domain 3 of the C. elegans EF-Tu is no longer adaptive to conventional aa-tRNA. In
fact, the residues in domain 3 that are involved in T-stem binding are not well conserved in the C. elegans EF-Tu (Fig. 1).
Taken together, these experimental results demonstrate that the
N-terminal domains (domains 1 and 2) of the mammalian and C. elegans mt EF-Tus, but not those of the eubacterial EF-Tu, could
be functionally equivalent in binding to the nematode T arm-lacking
aa-tRNA and that the C-terminal domains (domains 3 and 3') of nematode
mt EF-Tu are crucial for binding to this type of tRNA (see
"Discussion").
Whether or not a unique EF-Tu exists in nematode mitochondria has
long been an intriguing question, because conventional EF-Tus so far
elucidated recognize the tRNA T-stem, Two cases in which unique EF-Tus with C-terminal extensions are used
for translation are already known: SelB in some bacteria and its
equivalents in mammalian systems (43, 44) and EF-Tu in mammalian
mitochondria (27). The former is specific for selenocysteinyl-tRNA, which is involved in decoding a particular UGA stop codon by
recognizing both the tRNA and the stem-loop structure of mRNA
occurring just behind the UGA codon in bacterial systems. The long
C-terminal extension (domain 4) with about 250 amino acid residues is
considered to be involved in the recognition of the stem-loop structure
(43). We attempted to align the C. elegans mt EF-Tu domain
3' sequence with the SelB domain 4 sequence in some bacteria, but no
appreciable homology was observed, suggesting that the function of
domain 3' in nematode mt EF-Tu is different from domain 4 in SelB.
C-terminal extensions comprising 11 amino acid residues observed in
mammalian mt EF-Tus (Fig. 1) (27, 45, 46) may possess a role in
compensating for some incomplete tertiary structures of mammalian mt
tRNAs, such as a lack of tertiary interaction between the T and D arms at the elbow region or the presence of a slightly shorter T stem (1,
47), a possibility supported by the fact that bacterial EF-Tu
possessing no such extension binds to mammalian mt tRNAs less
efficiently than mammalian mt EF-Tu
(48).3 Domains 1 and 2 of
mammalian mt EF-Tu may also contribute to such compensation (discussed
below). However, because no homology is observed between this extension
and domain 3' of the C. elegans mt EF-Tu, the nematode
domain 3' function also seems to differ from that of the C-terminal
extension of mammalian mt EF-Tu.
With regard to the other domains, there is considerable homology among
amino acid residues in both domains 1 and 2 between the nematode and
the other mt EF-Tus, whereas the residues in domain 3 are quite
different. The residues in contact with GTP (colored orange
in Fig. 1) (49-52), the acceptor stem (blue), and the amino
acid moiety of aa-tRNA (yellow) (9) as well as those involved in the pocket of the amino acid side chain of aa-tRNA (green) (8), all of which are dispersed through domains 1 and 2, are highly conserved among these EF-Tus. The residues
characteristic for mt EF-Tus (purple) (41) are also well
conserved. Only the residues in contact with the T stem, which are
located in domain 3 (red) (9), are quite different in the
nematode mt EF-Tu compared with the other mt EF-Tus, which is quite
reasonable considering that nematode mt EF-Tu has no target T stem in
the mt tRNA (see below) and lost binding ability to conventional tRNA
having a T stem (Fig. 3B).
The results presented here strongly suggest that the immunodetectable
protein (Fig. 2) is actually the nematode mt EF-Tu, which, as shown in
Fig. 3, specifically recognizes T stem-lacking tRNA. To substantiate
the contention that the protein is an EF-Tu, we also attempted
poly(U)-dependent poly(Phe) synthesis in vitro using the protein. Although the nematode mt EF-Tu was found not to work
in an E. coli system, it only slightly stimulated
poly(U)-dependent poly(Phe) synthesis using E. coli Phe-tRNAPhe in a bovine in vitro
translation system (data not shown), despite very low affinities of the
nematode mt EF-Tu toward E. coli Phe-tRNAPhe.
Nematode mt Phe-tRNAPhe functioned neither on bovine mt
ribosomes nor on E. coli ribosomes. A homologous nematode mt
translation system, which is as yet unavailable, is necessary to detect
efficient translation activity with nematode mt EF-Tu.
The experimental results with the chimeric proteins (Fig. 4) indicate
that, in addition to domain 3', domain 3 in the nematode protein also
plays a crucial role in its binding to T arm-lacking tRNA (Fig.
5A), because the binding activity of bovine mt EF-Tu with
domain 3' of nematode mt EF-Tu attached (BmCe3') toward nematode mt
aa-tRNA was significantly enhanced by switching domain 3 between the
two animal mt EF-Tus. Domain 3 may fix the location of domain 3'
through interactions between the C-terminal domains so that domain 3'
can bind to the T arm-lacking tRNA at the proper position. Domain 3'
might be a part of the domain 3, which is larger than those of
conventional EF-Tus. In addition to the C-terminal domains, the
N-terminal domain 1 or both domains 1 and 2 in animal mt EF-Tus also
seem to assist in binding to the nematode mt T stem-lacking tRNA (Fig.
5A) or even to bovine mt tRNA with divergent T
arms.3
Although domain 1 is highly conserved among organisms, a key difference
between the peptide sequences in bacterial and mt EF-Tus lies in a
region noted previously as the "proteobacterial/mitochondrial factor
signature region" (41), which in mt EF-Tus is located near the
junction with domain 2 (colored purple in Fig. 1) but which
is absent in bacterial counterparts. In the case of animal mt factors,
this region might make a contribution in binding to the nematode mt
tRNAs, because, as shown in Fig. 5A, nematode Met-tRNAMet can be recognized not only by nematode mt EF-Tu
but also by bovine mt EF-Tu, although only slightly, whereas it is not
recognized at all by T. thermophilus EF-Tu.
The above results taken together with crystallographic data on the
bacterial ternary complex (8, 9) and bovine mt EF-Tu/GDP (46) prompt us
to propose the outline ternary complex model for nematode mitochondria
shown in Fig. 6 (right panel).
Because the C terminus of the eubacterial EF-Tu in the ternary complex is located near the connector region of tRNA (Fig. 6, left
panel) and it has been suggested that the short C-terminal
extension of bovine mt EF-Tu touches the connector region (46), domain 3' of the nematode mt EF-Tu may play a role in compensating for the
loss of interaction between the tRNA T stem and domain 3 of the
bacterial EF-Tu, either by binding domain 3' to a specific region(s)
around the connector region of the tRNA or by providing a new binding
site for the tRNA with the help of another EF-Tu domain(s). Although
the location of domain 3' in the complex is unknown, taking account of
the macromolecular mimicry hypothesis concerning translation machinery
(8, 53), we postulate that domain 3' in the nematode mt complex might
be located at or near the position corresponding to that occupied by
the T arm in the bacterial ternary complex (Fig. 6).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in eukaryotic cytoplasm (12,
13). On the other hand, most tRNA species encoded in the mt DNA of at
least three nematodes, Ascaris suum, Caenorhabditis
elegans, and Onchocerca volvulus, lack the T stem
necessary for binding with bacterial EF-Tu (3, 4, 14-19). (The tRNAs
having the T arm also exist in nematode mitochondria (15, 17, 18), and
the recognition of such tRNAs by another EF-Tu is now being studied in
our laboratory.) Nevertheless, it has been demonstrated that such
nematode mt tRNAs are capable of accepting the cognate amino acids and
are probably functional in the mt translation systems (3, 4, 20). To
elucidate how nematode mt EF-Tu binds to T stem-lacking tRNA, we
determined the full-length cDNA sequence of a nematode (C. elegans) EF-Tu homologue and detected the corresponding protein in
C. elegans mt extracts immunologically. Based on the binding
activity of the nematode EF-Tu homologue toward nematode and bacterial
aa-tRNAs revealed by our experimental results, we propose a structural model for the ternary complex formed among EF-Tu, aa-tRNA, and GTP in
the nematode mt system and postulate that the RNA co-evolved in the
system with its cognate RNA-binding protein.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-32P]dATP or [
-35S]dATPaS (Amersham
Pharmacia Biotech) and vector-specific primers (22, 23).
Oligodeoxynucleotide primers used in the study
cDNA clones were excised in vivo into the plasmid form
in the "popout" strain according to the protocol of the supplier
and then used for sequencing analysis. 5'-RACE using the
cDNA-specific primer 5'-RACE (Table I) was performed as described
(25). The cDNA clones were sequenced on automated DNA sequencers
(DSQ-1, Shimadzu; 373A, Applied Biosystems) using commercially
available kits (Perkin-Elmer; Amersham Pharmacia Biotech; Wakunaga;
Takara Shuzo).
-D-galactopyranoside and ampicillin were then added to the culture medium to give final concentrations of 1 mM and 500 µg/ml, respectively, and the cultivation was
continued at 18 °C overnight. The cells were harvested by
centrifugation and frozen until use. Cells were lysed by sonication and
fractionated according to Woriax et al. (27) with minor
modifications. For immunization, the putative mature form of the
C. elegans EF-Tu homologue with the tag sequence was used
after desalting by dialysis against 20 mM Hepes-KOH (pH
7.0), 40 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, and 10% (v/v) glycerol followed by concentration
with Centricon 10 (Amicon). For functional analysis, the proteins were
treated with biotinylated thrombin (Novagen) during dialysis to remove the tag sequence and purified by streptavidin-agarose (Life
Technologies, Inc.) column chromatography.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid alignment of EF-Tus. The
EF-Tu sequences of C. elegans (this study) (C. el, accession number D38471), human (X84694), bovine (L38996),
Arabidopsis thaliana (A. th, X89227), and yeast
(Saccaromyces cerevisiae) (K00428) mitochondria and of
E. coli (E. co, P02990), Thermus
aquaticus (T. aq, S29293), and T. thermophilus (T. th, P07157). The alignment was carried
out using the Clustal V program (54) followed by manual modifications.
* and , conserved and similar residues among EF-Tus shown here;
purple (abbreviated by p, which is shown at the
top of the sequences), mt EF-Tu-specific residues (41);
green (g), residues involved in the pocket of the
side chain of the aminoacyl group (8); yellow
(y), residues in contact with aminoacyl group (9);
blue (b), residues in contact with the acceptor
stem (9); red (r), residues in contact with the
T-stem (9); orange (o), residues involved in
guanine nucleotide binding (49-52). The transit peptide of bovine mt
EF-Tu (27) is shown by lowercase letters. The N-terminal
residue of each recombinant protein is indicated by a bold
letter. Boxed letters are random coiled regions around
domain junctions in bovine mt EF-Tu (46) and T. aquaticus
EF-Tu (8, 9). Filled and open triangles
show the borders between bovine mt and C. elegans mt
sequences of BmCe variants and those between T. thermophilus
and C. elegans mt sequences of TthCe variants,
respectively.
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Fig. 2.
Immunological detection of the EF-Tu
homologue from C. elegans by Western blotting.
Lane 1, recombinant mature EF-Tu of C. elegans
with the N-terminal His tag and linker sequences (52.6 kDa; 0.1 µg of
protein was loaded on the gel). Lane 2, mt fraction of
C. elegans (25 µg of protein). Lane 3,
cytoplasmic (cyt) fraction of C. elegans (17 µg
of protein).
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Fig. 3.
Binding activity of nematode mt and E. coli EF-Tus toward nematode mt and E. coli
Met-tRNAs. Deacylation-protection assay of the aminoacyl
ester bond against hydrolysis (34) of A. suum mt
Met-tRNAMet (A; initial concentration of
Met-tRNA, 33 nM) and E. coli elongator
Met-tRNAmMet (B, initial
concentration of Met-tRNA, 30 nM) in the presence of the
C. elegans mt EF-Tu or E. coli EF-Tu. , 1. 2 µM C. elegans protein;
, 3 µM
C. elegans protein;
, 1. 2 µM E.
coli EF-Tu;
, no protein.
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Fig. 4.
Schematic representations of chimeric EF-Tu
variants. All of the EF-Tu variants contain an additional sequence
(MGSSHHHHHHSSGLVPRGSHM) including the His tag and thrombin site
(LVPR GS) at the N terminus. The EF-Tu domains of C. elegans mitochondria are shown by white letters on a
black background, those of bovine mitochondria are shown by
black letters on a gray background, and those of
T. thermophilus are shown by black letters on a
white background. The open and filled
triangles are the borders between the different EF-Tus, as defined
in Fig. 1 legend.
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Fig. 5.
Deacylation-protection assay of A. suum mt Met-tRNAMet (A and
B) or E. coli
Met-tRNAmMet (C and
D) using chimeric EF-Tus (BmCe variants (A
and C) or TthCe variants (B and
D)). The amounts of Met-tRNAs remaining after 40 min of incubation at 30 °C with different concentrations of EF-Tus
are shown by percentages of the original amounts of Met-tRNAs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
which is absent in most
nematode mt tRNAs (2). The findings of this study could have given the
answer to this question by demonstrating that a nematode mt translation
system actually possesses a unique EF-Tu with a long C-terminal
extension (domain 3') (Figs. 1 and 2), which is considered to be a
prerequisite to compensate for the lack of a T stem in nematode mt
tRNAs (Fig. 3).
View larger version (36K):
[in a new window]
Fig. 6.
Schematic representations of a ternary
complex of bacterial EF-Tu, aa-tRNA, and GTP and a predicted model of
its nematode mt counterpart. Left panel, the bacterial
ternary complex (8, 9); right panel, the model of nematode
mt ternary complex. GTP is omitted. The EF-Tu is shown as a set of
shaded circles, each of which corresponds to the domains
numbered in order. The tRNAs are portrayed as simplified backbones with
the aminoacyl moiety at the 3' terminus depicted by gray filled
circles. Domains 1 and 2 of nematode mt EF-Tu are not homologous
with those of bacterial EF-Tu but similar to those of bovine mt EF-Tu
in terms of tRNA binding assay (Fig. 5). Domains 3 and 3' of the
nematode mt EF-Tu are unique regions for T arm-lacking tRNAs.
Finally, our results may represent a novel example of the co-evolution
of a structurally simplified RNA and the cognate RNA-binding protein,
with the latter having apparently acquired an additional domain to
compensate for the lack of a protein-binding site(s) in the RNA molecule.
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ACKNOWLEDGEMENTS |
---|
We thank J. E. Sulston, A. Coulson, and L. Fulton for the C. elegans cDNA clones, Y. Kohara (National Institute of Genetics, Japan) for information on genomic mapping of the cDNA, T. Horie, H. Suzuki, Y. Osada, T. Kikuchi, and T. Furuta (The University of Tokyo) for producing the antibody, L. L. Spremulli, T. Yokogawa, and N. Hayashi for materials, T. R. Unnasch (University of Alabama at Birmingham) for information on the Onchocerca mt DNA sequence data, and Shimadzu Corp. (Japan) for an automated DNA sequencer.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan (to K. K., S. K., and K. W.) and grants from the Japan Society for the Promotion of Science (to Y. W. and T. O.).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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D38471 and D38472.
b Present address: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
c Present address: RIKEN Inst., Kanagawa, Japan.
d Present address: Dept. of Industrial Chemistry, Chiba Institute of Technology, Chiba, Japan.
e Present address: Dept. of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan.
g Present address: School of Health Science, International University of Health and Welfare, Tochigi, Japan.
j To whom correspondence should be addressed: Dept. of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 113-8656, Japan. Tel. and Fax: 81-3-5800-6950; E-mail: kw@kwl.t.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M011118200
2 Y. Watanabe, K. Kita, and K. Watanabe, unpublished results.
3 T. Ohtsuki and K. Watanabe, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: mt, mitochondrial; EF-Tu, translation elongation factor Tu; aa-tRNA, aminoacyl-tRNA; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction.
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