An "Elongated" Translation Elongation Factor Tu for Truncated tRNAs in Nematode Mitochondria*

Takashi Ohtsukia, Yoh-ichi Watanabeab, Chie Takemotoac, Gota Kawaiad, Takuya Uedaae, Kiyoshi Kitabf, Somei Kojimafg, Yoshito Kaziroh, Jens Nyborgi, and Kimitsuna Watanabeaej

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 1alpha 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
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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 [alpha -32P]dATP or [alpha -35S]dATPaS (Amersham Pharmacia Biotech) and vector-specific primers (22, 23).

                              
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Table I
Oligodeoxynucleotide primers used in the study
Restriction enzyme sites are printed in bold type.

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 lambda  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).

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-beta -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.


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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).

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.


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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. triangle , 1. 2 µM C. elegans protein; black-triangle, 3 µM C. elegans protein; black-square, 1. 2 µM E. coli EF-Tu; open circle , no protein.

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.


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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 (LVPRdown-arrow 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.

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.


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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.

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").

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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,- 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).

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).


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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.

    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.

    FOOTNOTES

* 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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Sprinzl, M., Horn, C., Brown, M., Loudovitch, A., and Steinberg, S. (1998) Nucleic Acids Res. 26, 148-153[Abstract/Free Full Text]
2. Wolstenholme, D. R. (1992) Int. Rev. Cytol. 141, 173-216[Medline] [Order article via Infotrieve]
3. Watanabe, Y., Tsurui, H., Ueda, T., Furushima, R., Takamiya, S., Kita, K., Nishikawa, K., and Watanabe, K. (1994) J. Biol. Chem. 269, 22902-22906[Abstract/Free Full Text]
4. Watanabe, Y., Tsurui, H., Ueda, T., Furushima-Shimogawara, R., Takamiya, S., Kita, K., Nishikawa, K., and Watanabe, K. (1997) Biochim. Biophys. Acta 1350, 119-122[Medline] [Order article via Infotrieve]
5. Palmer, J. D. (1997) Nature 387, 454-455[Medline] [Order article via Infotrieve]
6. Kaziro, Y. (1978) Biochim. Biophys. Acta 505, 95-127[Medline] [Order article via Infotrieve]
7. Sprinzl, M. (1994) Trends Biochem. Sci. 19, 245-250[CrossRef][Medline] [Order article via Infotrieve]
8. Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B. F., and Nyborg, J. (1995) Science 270, 1464-1472[Abstract]
9. Nissen, P., Thirup, S., Kjeldgaard, M., and Nyborg, J. (1999) Structure 7, 143-156[CrossRef][Medline] [Order article via Infotrieve]
10. Clark, B. F. C., Kjeldgaard, M., Barciszewski, J., and Sprinzl, M. (1995) in tRNA: Structure, Biosynthesis and Function (Söll, D. , and RajBahndary, U. L., eds) , pp. 423-442, American Society for Microbiology, Washington, D.C.
11. Rudinger, J., Blechschmidt, B., Ribeiro, S., and Sprinzl, M. (1994) Biochemistry 33, 5682-5688[Medline] [Order article via Infotrieve]
12. Forster, C., Chakraburtty, K., and Sprinzl, M. (1993) Nucleic Acids Res. 21, 5679-5683[Abstract]
13. Dreher, T. W., Uhlenbeck, O. C., and Browning, K. S. (1999) J. Biol. Chem. 274, 666-672[Abstract/Free Full Text]
14. Wolstenholme, D. R., Macfarlane, J. L., Okimoto, R., Clary, D. O., and Wahleithner, J. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1324-1328[Abstract]
15. Okimoto, R., and Wolstenholme, D. R. (1990) EMBO J. 9, 3405-3411[Abstract]
16. Okimoto, R., Macfarlane, J. L., Clary, D. O., and Wolstenholme, D. R. (1992) Genetics 130, 471-498[Abstract/Free Full Text]
17. Wolstenholme, D. R., Okimoto, R., and Macfarlane, J. L. (1994) Nucleic Acids Res. 22, 4300-4306[Abstract]
18. Keddie, E. M., Higazi, T., and Unnasch, T. R. (1998) Mol. Biochem. Parasitol. 95, 111-27[CrossRef][Medline] [Order article via Infotrieve]
19. Ohtsuki, T., Kawai, G., and Watanabe, K. (1998) J. Biochem. (Tokyo) 124, 28-34[Abstract]
20. Ohtsuki, T., Kawai, G., Watanabe, Y., Kita, K., Nishikawa, K., and Watanabe, K. (1996) Nucleic Acids Res. 24, 662-667[Abstract/Free Full Text]
21. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 33, 103-119[CrossRef][Medline] [Order article via Infotrieve]
22. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract]
23. Tabor, S., and Richardson, C. C. (1989) J. Biol. Chem. 264, 6447-6458[Abstract/Free Full Text]
24. Waterston, R., Martin, C., Craxton, M., Huynh, C., Coulson, A., Hillier, L., Durbin, R., Green, P., Shownkeen, R., Halloran, N., Metzstein, M., Hawkins, T., Wilson, R., Berks, M., Du, Z., Thomas, K., Thierry-Mieg, J., and Sulston, J. (1992) Nat. Genet. 1, 114-123[Medline] [Order article via Infotrieve]
25. Watanabe, Y., Kita, K., Ueda, T., and Watanabe, K. (1997) Biochim. Biophys. Acta 1353, 7-12[Medline] [Order article via Infotrieve]
26. Ahmadian, M. R., Kreutzer, R., and Sprinzl, M. (1991) Biochimie (Paris) 73, 1037-1043[CrossRef][Medline] [Order article via Infotrieve]
27. Woriax, V. L., Burkhart, W., and Spremulli, L. L. (1995) Biochim. Biophys. Acta 1264, 347-356[Medline] [Order article via Infotrieve]
28. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89[Medline] [Order article via Infotrieve]
29. Tartof, K. D., and Hobbs, C. A. (1987) Bethesda Res. Lab. Focus 9, 12
30. Roth, J., Lentze, M. J., and Berger, E. G. (1985) J. Cell Biol. 100, 113-125
31. Kuramochi, T., Hirawake, H., Kojima, S., Takamiya, S., Furushima, R., Aoki, T., Komuniecki, R., and Kita, K. (1994) Mol. Biochem. Parasitol. 68, 177-187[Medline] [Order article via Infotrieve]
32. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
33. Saruta, F., Kuramochi, T., Nakamura, K., Takamiya, S., Yu, Y., Aoki, T., Sekimizu, K., Kojima, S., and Kita, K. (1995) J. Biol. Chem. 270, 928-932[Abstract/Free Full Text]
34. Takemoto, C., Koike, T., Yokogawa, T., Benkowski, L., Spremulli, L. L., Ueda, T., Nishikawa, K., and Watanabe, K. (1995) Biochimie (Paris) 77, 104-108[CrossRef][Medline] [Order article via Infotrieve]
35. Jacobson, G. R., and Rosenbusch, J. P. (1977) FEBS Lett. 79, 8-10[CrossRef][Medline] [Order article via Infotrieve]
36. Pingoud, A., Urbanke, C., Krauss, G., Peters, F., and Maass, G. (1977) Eur. J. Biochem. 78, 403-409[Abstract]
37. Tabara, H., Motohashi, T., and Kohara, Y. (1996) Nucleic Acids Res. 24, 2119-2124[Abstract/Free Full Text]
38. Krause, M., and Hirsh, D. (1987) Cell 49, 753-761[Medline] [Order article via Infotrieve]
39. Hartl, F. U., Pfanner, N., Nicholson, D., and Neupert, W. (1989) Biochim. Biophys. Acta 988, 1-45[Medline] [Order article via Infotrieve]
40. Hendrick, J. P., Hodges, P. E., and Rosenberg, L. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4056-4060[Abstract]
41. Kuhlman, P., and Palmer, J. D. (1995) Mol. Biol. 29, 1057-1070
42. Bullard, J. M., Cai, Y.-C., Zhang, Y., and Spremulli, L. L. (1999) Biochim. Biophys. Acta 1446, 102-114[Medline] [Order article via Infotrieve]
43. Kromayer, M., Wilting, R., Tormay, P., and Böck, A. (1996) J. Mol. Biol. 262, 413-420[CrossRef][Medline] [Order article via Infotrieve]
44. Fagegaltier, D., Hubert, N., Yamada, K., Mizutani, T., Carbon, P., and Krol, A. (2000) EMBO J. 19, 4796-4805[Abstract/Free Full Text]
45. Ling, M., Merante, F., Chen, H. S., Duff, C., Duncan, A. M., and Robinson, B. H. (1997) Gene (Amst.) 197, 325-336[CrossRef][Medline] [Order article via Infotrieve]
46. Andersen, G. R., Thirup, S., Spremulli, L. L., and Nyborg, J. (2000) J. Mol. Biol. 297, 421-436[CrossRef][Medline] [Order article via Infotrieve]
47. Wakita, K., Watanabe, Y., Yokogawa, T., Kumazawa, Y., Nakamura, S., Ueda, T., Watanabe, K., and Nishikawa, K. (1994) Nucleic Acids Res. 22, 347-353[Abstract]
48. Kumazawa, Y., Schwartzbach, C. J., Liao, H. X., Mizumoto, K., Kaziro, Y., Miura, K., Watanabe, K., and Spremulli, L. L. (1991) Biochim. Biophys. Acta 1090, 167-172[Medline] [Order article via Infotrieve]
49. Kjeldgaard, M., and Nyborg, J. (1992) J. Mol. Biol. 223, 721-742[Medline] [Order article via Infotrieve]
50. Berchtold, H., Reshetnikova, L., Reiser, C. O., Schirmer, N. K., Sprinzl, M., and Hilgenfeld, R. (1993) Nature 365, 126-132[CrossRef][Medline] [Order article via Infotrieve]
51. Kjeldgaard, M., Nissen, P., Thirup, S., and Nyborg, J. (1993) Structure 1, 35-50[Medline] [Order article via Infotrieve]
52. Polekhina, G., Thirup, S., Kjeldgaard, M., Nissen, P., Lippmann, C., and Nyborg, J. (1996) Structure 4, 1141-1151[Medline] [Order article via Infotrieve]
53. Ito, K., Ebihara, K., Uno, M., and Nakamura, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5443-5448[Abstract/Free Full Text]
54. Higgins, D. G., Bleasby, A. J., and Fuchs, R. (1991) Comput. Appl. Biosci. 8, 189-191[Abstract]


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