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W2031824657 abstract "We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematodeCaenorhabditis 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.D38471D38472 We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematodeCaenorhabditis 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.D38471D38472 mitochondrial translation elongation factor Tu aminoacyl-tRNA rapid amplification of cDNA ends polymerase chain reaction One of the unusual features of the animal mitochondrial (mt)1 translation system is the use of tRNA with highly divergent structures (1Sprinzl M. Horn C. Brown M. Loudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (806) Google Scholar). Such nonstandard structural forms have been predicted from gene sequences encoded in mt DNA (2Wolstenholme D.R. Int. Rev. Cytol. 1992; 141: 173-216Crossref PubMed Scopus (1251) Google Scholar), and some have been confirmed at the RNA level (3Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar, 4Watanabe Y. Tsurui H. Ueda T. Furushima-Shimogawara R. Takamiya S. Kita K. Nishikawa K. Watanabe K. Biochim. Biophys. Acta. 1997; 1350: 119-122Crossref PubMed Scopus (21) Google Scholar). Mitochondrial tRNA structural divergence is thought to reflect relaxed constraints operating on translation systems that produce very few proteins encoded in mt DNA (5Palmer J.D. Nature. 1997; 387: 454-455Crossref PubMed Scopus (35) Google Scholar). 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 (6Kaziro Y. Biochim. Biophys. Acta. 1978; 505: 95-127Crossref PubMed Scopus (398) Google Scholar, 7Sprinzl M. Trends Biochem. Sci. 1994; 19: 245-250Abstract Full Text PDF PubMed Scopus (81) Google Scholar). Recent crystallographic analysis (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) as well as a variety of biochemical data (10Clark B.F.C. Kjeldgaard M. Barciszewski J. Sprinzl M. Söll D. RajBahndary U.L. tRNA: Structure, Biosynthesis and Function. American Society for Microbiology, Washington, D.C.1995: 423-442Google Scholar) 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 (11Rudinger J. Blechschmidt B. Ribeiro S. Sprinzl M. Biochemistry. 1994; 33: 5682-5688Crossref PubMed Scopus (46) Google Scholar). Similar tRNA binding mechanisms have been proposed for elongation factor 1α in eukaryotic cytoplasm (12Forster C. Chakraburtty K. Sprinzl M. Nucleic Acids Res. 1993; 21: 5679-5683Crossref PubMed Scopus (33) Google Scholar,13Dreher T.W. Uhlenbeck O.C. Browning K.S. J. Biol. Chem. 1999; 274: 666-672Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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 (3Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar, 4Watanabe Y. Tsurui H. Ueda T. Furushima-Shimogawara R. Takamiya S. Kita K. Nishikawa K. Watanabe K. Biochim. Biophys. Acta. 1997; 1350: 119-122Crossref PubMed Scopus (21) Google Scholar, 14Wolstenholme D.R. Macfarlane J.L. Okimoto R. Clary D.O. Wahleithner J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1324-1328Crossref PubMed Scopus (210) Google Scholar, 15Okimoto R. Wolstenholme D.R. EMBO J. 1990; 9: 3405-3411Crossref PubMed Scopus (98) Google Scholar, 16Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar, 17Wolstenholme D.R. Okimoto R. Macfarlane J.L. Nucleic Acids Res. 1994; 22: 4300-4306Crossref PubMed Scopus (47) Google Scholar, 18Keddie E.M. Higazi T. Unnasch T.R. Mol. Biochem. Parasitol. 1998; 95: 27-111Crossref Scopus (142) Google Scholar, 19Ohtsuki T. Kawai G. Watanabe K. J. Biochem. ( Tokyo ). 1998; 124: 28-34Crossref PubMed Scopus (27) Google Scholar). (The tRNAs having the T arm also exist in nematode mitochondria (15Okimoto R. Wolstenholme D.R. EMBO J. 1990; 9: 3405-3411Crossref PubMed Scopus (98) Google Scholar, 17Wolstenholme D.R. Okimoto R. Macfarlane J.L. Nucleic Acids Res. 1994; 22: 4300-4306Crossref PubMed Scopus (47) Google Scholar, 18Keddie E.M. Higazi T. Unnasch T.R. Mol. Biochem. Parasitol. 1998; 95: 27-111Crossref Scopus (142) Google Scholar), 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 (3Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar, 4Watanabe Y. Tsurui H. Ueda T. Furushima-Shimogawara R. Takamiya S. Kita K. Nishikawa K. Watanabe K. Biochim. Biophys. Acta. 1997; 1350: 119-122Crossref PubMed Scopus (21) Google Scholar, 20Ohtsuki T. Kawai G. Watanabe Y. Kita K. Nishikawa K. Watanabe K. Nucleic Acids Res. 1996; 24: 662-667Crossref PubMed Scopus (34) Google Scholar). 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 inC. 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.DISCUSSIONWhether 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 (2Wolstenholme D.R. Int. Rev. Cytol. 1992; 141: 173-216Crossref PubMed Scopus (1251) Google Scholar). 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 (43Kromayer M. Wilting R. Tormay P. Böck A. J. Mol. Biol. 1996; 262: 413-420Crossref PubMed Scopus (98) Google Scholar, 44Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (242) Google Scholar) and EF-Tu in mammalian mitochondria (27Woriax V.L. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1264: 347-356Crossref PubMed Scopus (77) Google Scholar). 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 (43Kromayer M. Wilting R. Tormay P. Böck A. J. Mol. Biol. 1996; 262: 413-420Crossref PubMed Scopus (98) Google Scholar). 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) (27Woriax V.L. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1264: 347-356Crossref PubMed Scopus (77) Google Scholar, 45Ling M. Merante F. Chen H.S. Duff C. Duncan A.M. Robinson B.H. Gene ( Amst. ). 1997; 197: 325-336Crossref PubMed Scopus (43) Google Scholar, 46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar) 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 (1Sprinzl M. Horn C. Brown M. Loudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (806) Google Scholar,47Wakita K. Watanabe Y. Yokogawa T. Kumazawa Y. Nakamura S. Ueda T. Watanabe K. Nishikawa K. Nucleic Acids Res. 1994; 22: 347-353Crossref PubMed Scopus (75) Google Scholar), 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 (48Kumazawa Y. Schwartzbach C.J. Liao H.X. Mizumoto K. Kaziro Y. Miura K. Watanabe K. Spremulli L.L. Biochim. Biophys. Acta. 1991; 1090: 167-172Crossref PubMed Scopus (38) Google Scholar). 3T. Ohtsuki and K. Watanabe, unpublished results. 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 orangein Fig. 1) (49Kjeldgaard M. Nyborg J. J. Mol. Biol. 1992; 223: 721-742Crossref PubMed Scopus (246) Google Scholar, 50Berchtold H. Reshetnikova L. Reiser C.O. Schirmer N.K. Sprinzl M. Hilgenfeld R. Nature. 1993; 365: 126-132Crossref PubMed Scopus (514) Google Scholar, 51Kjeldgaard M. Nissen P. Thirup S. Nyborg J. Structure. 1993; 1: 35-50Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 52Polekhina G. Thirup S. Kjeldgaard M. Nissen P. Lippmann C. Nyborg J. Structure. 1996; 4: 1141-1151Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), the acceptor stem (blue), and the amino acid moiety of aa-tRNA (yellow) (9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) as well as those involved in the pocket of the amino acid side chain of aa-tRNA (green) (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar), 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) (41Kuhlman P. Palmer J.D. Mol. Biol. 1995; 29: 1057-1070Crossref Scopus (18) Google Scholar) are also well conserved. Only the residues in contact with the T stem, which are located in domain 3 (red) (9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), 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. 3 B).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 vitrousing 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 vitrotranslation 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.5 A), 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.5 A) or even to bovine mt tRNA with divergent T arms.3Although 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” (41Kuhlman P. Palmer J.D. Mol. Biol. 1995; 29: 1057-1070Crossref Scopus (18) Google Scholar), 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. 5 A, 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 (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) and bovine mt EF-Tu/GDP (46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar) 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 (46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar), 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 (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 53Ito K. Ebihara K. Uno M. Nakamura Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5443-5448Crossref PubMed Scopus (128) Google Scholar), 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).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. One of the unusual features of the animal mitochondrial (mt)1 translation system is the use of tRNA with highly divergent structures (1Sprinzl M. Horn C. Brown M. Loudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (806) Google Scholar). Such nonstandard structural forms have been predicted from gene sequences encoded in mt DNA (2Wolstenholme D.R. Int. Rev. Cytol. 1992; 141: 173-216Crossref PubMed Scopus (1251) Google Scholar), and some have been confirmed at the RNA level (3Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar, 4Watanabe Y. Tsurui H. Ueda T. Furushima-Shimogawara R. Takamiya S. Kita K. Nishikawa K. Watanabe K. Biochim. Biophys. Acta. 1997; 1350: 119-122Crossref PubMed Scopus (21) Google Scholar). Mitochondrial tRNA structural divergence is thought to reflect relaxed constraints operating on translation systems that produce very few proteins encoded in mt DNA (5Palmer J.D. Nature. 1997; 387: 454-455Crossref PubMed Scopus (35) Google Scholar). 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 (6Kaziro Y. Biochim. Biophys. Acta. 1978; 505: 95-127Crossref PubMed Scopus (398) Google Scholar, 7Sprinzl M. Trends Biochem. Sci. 1994; 19: 245-250Abstract Full Text PDF PubMed Scopus (81) Google Scholar). Recent crystallographic analysis (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) as well as a variety of biochemical data (10Clark B.F.C. Kjeldgaard M. Barciszewski J. Sprinzl M. Söll D. RajBahndary U.L. tRNA: Structure, Biosynthesis and Function. American Society for Microbiology, Washington, D.C.1995: 423-442Google Scholar) 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 (11Rudinger J. Blechschmidt B. Ribeiro S. Sprinzl M. Biochemistry. 1994; 33: 5682-5688Crossref PubMed Scopus (46) Google Scholar). Similar tRNA binding mechanisms have been proposed for elongation factor 1α in eukaryotic cytoplasm (12Forster C. Chakraburtty K. Sprinzl M. Nucleic Acids Res. 1993; 21: 5679-5683Crossref PubMed Scopus (33) Google Scholar,13Dreher T.W. Uhlenbeck O.C. Browning K.S. J. Biol. Chem. 1999; 274: 666-672Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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 (3Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar, 4Watanabe Y. Tsurui H. Ueda T. Furushima-Shimogawara R. Takamiya S. Kita K. Nishikawa K. Watanabe K. Biochim. Biophys. Acta. 1997; 1350: 119-122Crossref PubMed Scopus (21) Google Scholar, 14Wolstenholme D.R. Macfarlane J.L. Okimoto R. Clary D.O. Wahleithner J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1324-1328Crossref PubMed Scopus (210) Google Scholar, 15Okimoto R. Wolstenholme D.R. EMBO J. 1990; 9: 3405-3411Crossref PubMed Scopus (98) Google Scholar, 16Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar, 17Wolstenholme D.R. Okimoto R. Macfarlane J.L. Nucleic Acids Res. 1994; 22: 4300-4306Crossref PubMed Scopus (47) Google Scholar, 18Keddie E.M. Higazi T. Unnasch T.R. Mol. Biochem. Parasitol. 1998; 95: 27-111Crossref Scopus (142) Google Scholar, 19Ohtsuki T. Kawai G. Watanabe K. J. Biochem. ( Tokyo ). 1998; 124: 28-34Crossref PubMed Scopus (27) Google Scholar). (The tRNAs having the T arm also exist in nematode mitochondria (15Okimoto R. Wolstenholme D.R. EMBO J. 1990; 9: 3405-3411Crossref PubMed Scopus (98) Google Scholar, 17Wolstenholme D.R. Okimoto R. Macfarlane J.L. Nucleic Acids Res. 1994; 22: 4300-4306Crossref PubMed Scopus (47) Google Scholar, 18Keddie E.M. Higazi T. Unnasch T.R. Mol. Biochem. Parasitol. 1998; 95: 27-111Crossref Scopus (142) Google Scholar), 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 (3Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar, 4Watanabe Y. Tsurui H. Ueda T. Furushima-Shimogawara R. Takamiya S. Kita K. Nishikawa K. Watanabe K. Biochim. Biophys. Acta. 1997; 1350: 119-122Crossref PubMed Scopus (21) Google Scholar, 20Ohtsuki T. Kawai G. Watanabe Y. Kita K. Nishikawa K. Watanabe K. Nucleic Acids Res. 1996; 24: 662-667Crossref PubMed Scopus (34) Google Scholar). 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 inC. 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. DISCUSSIONWhether 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 (2Wolstenholme D.R. Int. Rev. Cytol. 1992; 141: 173-216Crossref PubMed Scopus (1251) Google Scholar). 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 (43Kromayer M. Wilting R. Tormay P. Böck A. J. Mol. Biol. 1996; 262: 413-420Crossref PubMed Scopus (98) Google Scholar, 44Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (242) Google Scholar) and EF-Tu in mammalian mitochondria (27Woriax V.L. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1264: 347-356Crossref PubMed Scopus (77) Google Scholar). 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 (43Kromayer M. Wilting R. Tormay P. Böck A. J. Mol. Biol. 1996; 262: 413-420Crossref PubMed Scopus (98) Google Scholar). 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) (27Woriax V.L. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1264: 347-356Crossref PubMed Scopus (77) Google Scholar, 45Ling M. Merante F. Chen H.S. Duff C. Duncan A.M. Robinson B.H. Gene ( Amst. ). 1997; 197: 325-336Crossref PubMed Scopus (43) Google Scholar, 46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar) 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 (1Sprinzl M. Horn C. Brown M. Loudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (806) Google Scholar,47Wakita K. Watanabe Y. Yokogawa T. Kumazawa Y. Nakamura S. Ueda T. Watanabe K. Nishikawa K. Nucleic Acids Res. 1994; 22: 347-353Crossref PubMed Scopus (75) Google Scholar), 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 (48Kumazawa Y. Schwartzbach C.J. Liao H.X. Mizumoto K. Kaziro Y. Miura K. Watanabe K. Spremulli L.L. Biochim. Biophys. Acta. 1991; 1090: 167-172Crossref PubMed Scopus (38) Google Scholar). 3T. Ohtsuki and K. Watanabe, unpublished results. 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 orangein Fig. 1) (49Kjeldgaard M. Nyborg J. J. Mol. Biol. 1992; 223: 721-742Crossref PubMed Scopus (246) Google Scholar, 50Berchtold H. Reshetnikova L. Reiser C.O. Schirmer N.K. Sprinzl M. Hilgenfeld R. Nature. 1993; 365: 126-132Crossref PubMed Scopus (514) Google Scholar, 51Kjeldgaard M. Nissen P. Thirup S. Nyborg J. Structure. 1993; 1: 35-50Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 52Polekhina G. Thirup S. Kjeldgaard M. Nissen P. Lippmann C. Nyborg J. Structure. 1996; 4: 1141-1151Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), the acceptor stem (blue), and the amino acid moiety of aa-tRNA (yellow) (9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) as well as those involved in the pocket of the amino acid side chain of aa-tRNA (green) (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar), 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) (41Kuhlman P. Palmer J.D. Mol. Biol. 1995; 29: 1057-1070Crossref Scopus (18) Google Scholar) are also well conserved. Only the residues in contact with the T stem, which are located in domain 3 (red) (9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), 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. 3 B).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 vitrousing 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 vitrotranslation 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.5 A), 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.5 A) or even to bovine mt tRNA with divergent T arms.3Although 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” (41Kuhlman P. Palmer J.D. Mol. Biol. 1995; 29: 1057-1070Crossref Scopus (18) Google Scholar), 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. 5 A, 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 (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) and bovine mt EF-Tu/GDP (46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar) 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 (46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar), 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 (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 53Ito K. Ebihara K. Uno M. Nakamura Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5443-5448Crossref PubMed Scopus (128) Google Scholar), 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).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. 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 (2Wolstenholme D.R. Int. Rev. Cytol. 1992; 141: 173-216Crossref PubMed Scopus (1251) Google Scholar). 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 (43Kromayer M. Wilting R. Tormay P. Böck A. J. Mol. Biol. 1996; 262: 413-420Crossref PubMed Scopus (98) Google Scholar, 44Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (242) Google Scholar) and EF-Tu in mammalian mitochondria (27Woriax V.L. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1264: 347-356Crossref PubMed Scopus (77) Google Scholar). 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 (43Kromayer M. Wilting R. Tormay P. Böck A. J. Mol. Biol. 1996; 262: 413-420Crossref PubMed Scopus (98) Google Scholar). 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) (27Woriax V.L. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1264: 347-356Crossref PubMed Scopus (77) Google Scholar, 45Ling M. Merante F. Chen H.S. Duff C. Duncan A.M. Robinson B.H. Gene ( Amst. ). 1997; 197: 325-336Crossref PubMed Scopus (43) Google Scholar, 46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar) 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 (1Sprinzl M. Horn C. Brown M. Loudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (806) Google Scholar,47Wakita K. Watanabe Y. Yokogawa T. Kumazawa Y. Nakamura S. Ueda T. Watanabe K. Nishikawa K. Nucleic Acids Res. 1994; 22: 347-353Crossref PubMed Scopus (75) Google Scholar), 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 (48Kumazawa Y. Schwartzbach C.J. Liao H.X. Mizumoto K. Kaziro Y. Miura K. Watanabe K. Spremulli L.L. Biochim. Biophys. Acta. 1991; 1090: 167-172Crossref PubMed Scopus (38) Google Scholar). 3T. Ohtsuki and K. Watanabe, unpublished results. 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 orangein Fig. 1) (49Kjeldgaard M. Nyborg J. J. Mol. Biol. 1992; 223: 721-742Crossref PubMed Scopus (246) Google Scholar, 50Berchtold H. Reshetnikova L. Reiser C.O. Schirmer N.K. Sprinzl M. Hilgenfeld R. Nature. 1993; 365: 126-132Crossref PubMed Scopus (514) Google Scholar, 51Kjeldgaard M. Nissen P. Thirup S. Nyborg J. Structure. 1993; 1: 35-50Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 52Polekhina G. Thirup S. Kjeldgaard M. Nissen P. Lippmann C. Nyborg J. Structure. 1996; 4: 1141-1151Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), the acceptor stem (blue), and the amino acid moiety of aa-tRNA (yellow) (9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) as well as those involved in the pocket of the amino acid side chain of aa-tRNA (green) (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar), 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) (41Kuhlman P. Palmer J.D. Mol. Biol. 1995; 29: 1057-1070Crossref Scopus (18) Google Scholar) are also well conserved. Only the residues in contact with the T stem, which are located in domain 3 (red) (9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), 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. 3 B). 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 vitrousing 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 vitrotranslation 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.5 A), 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.5 A) 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” (41Kuhlman P. Palmer J.D. Mol. Biol. 1995; 29: 1057-1070Crossref Scopus (18) Google Scholar), 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. 5 A, 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 (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 9Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Structure. 1999; 7: 143-156Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) and bovine mt EF-Tu/GDP (46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar) 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 (46Andersen G.R. Thirup S. Spremulli L.L. Nyborg J. J. Mol. Biol. 2000; 297: 421-436Crossref PubMed Scopus (53) Google Scholar), 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 (8Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (797) Google Scholar, 53Ito K. Ebihara K. Uno M. Nakamura Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5443-5448Crossref PubMed Scopus (128) Google Scholar), 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). 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. 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 theOnchocerca mt DNA sequence data, and Shimadzu Corp. (Japan) for an automated DNA sequencer." @default.
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W2031824657 title "An “Elongated” Translation Elongation Factor Tu for Truncated tRNAs in Nematode Mitochondria" @default.
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