Ohtsuki T et al. (2002) Nucleic Acids Res "Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu."

Ohtsuki T, Sato A, Sakurai M, Watanabe K

[Nucleic Acids Res, 2002]

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different

Sato A et al. (2006) Biochemistry "Identification of the residues involved in the unique serine specificity of Caenorhabditis elegans mitochondrial EF-Tu2."

Suzuki T, Watanabe K, Ohtsuki T, Sato A, Komiyama M, Watanabe YI

[Biochemistry, 2006]

In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.

Suematsu T et al. (2005) Nucleic Acids Res "A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2."

Ohtsuki T, Watanabe K, Sato A, Suematsu T, Sakurai M

[Nucleic Acids Res, 2005]

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA(Ser) that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D-T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.

Sakurai M et al. (2006) Biochem J "A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria."

Watanabe K, Watanabe YI, Sakurai M, Ohtsuki T

[Biochem J, 2006]

Nematode mitochondria possess extremely truncated tRNAs. Twenty of 22 tRNAs lack the entire T-arm. The T arm is necessary for the binding of canonical tRNAs and elongation factor (EF)-Tu. The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.

Ohtsuki T et al. (2002) Nat Struct Biol "A unique serine-specific elongation factor Tu found in nematode mitochondria."

Watanabe K, Watanabe Y, Sato A, Ohtsuki T

[Nat Struct Biol, 2002]

The translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to ribosomes by recognizing the tRNA acceptor and T stems. However, the unusual truncation observed in some animal mitochondrial tRNAs seems to prevent recognition by a canonical EF-Tu. For instance, nematode mitochondria contain tRNAs lacking a T or D arm. We recently found an atypical EF-Tu (EF-Tu1) specific for nematode mitochondrial tRNAs that lack the T arm. We have now discovered a second factor, EF-Tu2, which binds only to tRNAs that lack a D arm. EF-Tu2 seems unique in its amino acid specificity because it recognizes the aminoacyl moiety of seryl-tRNAs and the tRNA structure itself. Such EF-Tu evolution might explain tRNA structural divergence in animal mitochondria.

Kimata Y et al. (1994) J Biol Chem "Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth."

Kohno K, Kimata Y

[J Biol Chem, 1994]

Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains an unusual modified histidine residue, termed diphthamide. Diphthamide has been shown to be a site of ADP-ribosylation by bacterial toxins, but its function remains obscure. We expressed mutant genes of EF-2 with substitutions of 19 other amino acids for His-699, which is modified to diphthamide, in yeast cells and found that they can be classified into three groups. In the first group (Group 1), replacement of His-699 by the basic amino acid Arg or Lys showed not only loss of EF-2 activity but also inhibitory effects on the growth of cells co-expressing wild-type EF-2. In the second group (Group 2), replacement with Gly, Pro, Ser, or Asp resulted in nonfunctional EF-2, but it did not affect the growth of cells co-expressing wild-type EF-2. In the third group (Group 3), replacement by one of the other 13 amino acids resulted in a functional EF-2. In the Group 3 mutants, EF-2 was not ribosylated by diphtheria toxin, indicating that the mutant EF-2s did not form diphthamide. However, the viable cells grew more slowly than cells expressing wild-type EF-2 and showed temperature sensitivities. This result suggests that diphthamide may confer heat resistance on EF-2, although it still may be active without diphthamide at a normal temperature.

N Tavernarakis et al. (1999) International C. elegans Meeting "Knockout of Elongation Factor-2 Kinase Extends Lifespan in C. elegans"

AG Ryazanov, L Zhang, CE Mendola, M Driscoll, KS Pavur, Z Altun-Gultekin, W Wadsworth, N Tavernarakis

[International C. elegans Meeting, 1999]

Elongation factor-2 kinase (EF-2 kinase) is a ubiquitous protein kinase that regulates protein synthesis by phosphorylating and inactivating elongation factor-2 (EF-2), which catalyzes movement of the ribosome along mRNA during translation (Nature 334:170-173). EF-2 kinase belongs to an emerging new class of protein kinases called alpha-kinases that are structurally and evolutionarily unrelated to conventional eukaryotic protein kinases (Proc. Natl. Acad. Sci. 94: 4884-4889; Curr. Biol. 9: R43-R45). The physiological role of EF-2 phosphorylation remains a mystery. To investigate the role of EF-2 kinase, we isolated a TC1 insertion mutant for C. elegans EF-2 kinase. The TC1 insertion is in the middle of exon 2 which contains the EF-2 kinase catalytic domain. The TC1 insertion completely abolishes EF-2 kinase activity. Mutant worms lacking functional EF-2 kinase showed slightly decreased locomotion, pharyngeal pumping, defecation, and developmental rates. The mutant phenotypes were rescued by injection of the wild-type EF-2 kinase gene. Intriguingly, C. elegans eEF-2 kinase loss of function mutants live 25% longer than do wild type animals. These results provide the first direct evidence that regulation of protein synthesis and longevity may be intimately related.

Ohtsuki T et al. (2007) IUBMB Life "T-armless tRNAs and elongated elongation factor Tu."

Ohtsuki T, Watanabe Y

[IUBMB Life, 2007]

Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins. IUBMB Life, 59: 68-75, 2007.

Kjeldgaard M (2002) Structure "Another worm in translation."

Kjeldgaard M

[Structure, 2002]

Elongation factor EF-Tu is a key component in the translation step of protein synthesis, where it forms a complex with amino-acyl tRNA and delivers it to the ribosome. Until now, none of the known EF-Tu molecules have discriminated between the different species of tRNA, but now a new discovery sheds light on a curious EF-Tu homolog that binds just a single tRNA species.

Cai WJ et al. (2008) Zhongguo Zhong Xi Yi Jie He Za Zhi "[Effect of Epimedium flavonoids in retarding aging of C. elegans]"

Huang JH, Cai WJ, Zhang XM

[Zhongguo Zhong Xi Yi Jie He Za Zhi, 2008]

OBJECTIVE: To investigate effect of Epimedium flavonoids (EF), positively controlled by caloric restriction (CR) method, in retarding aging of the model organism C. elegans, in order to establish a basis for studying its action mechanism. METHODS: Experiment for life-time analysis was conducted on animals grouped into the blank group, the CR group, and the high and low dose EF groups to observe their mean lifespan, maximum lifespan and age-dependent mortality. And the reproductive capacity test and acute heat-stress analysis were carried out in the blank group and the high dose EF group to observe the subalgebra and the mean survival time under acute heat-stress at 35 degrees C. RESULTS: As compared with the blank group, the mean lifespan in the two EF group and the maximum lifespan in the high dose EF group were higher, and the age-dependent mortality in the high dose EF group was lower significantly (P<0.05 or P<0.01); as compared with the CR group, the mean lifespan and maximum lifespan in the high dose EF group were higher (P<0.01); but no significant difference of the subalgebra between the blank group and the high dose EF group was shown (P>0.05). Compared with the blank group, the mean lifespan in the high dose EF group was significantly prolonged under acute heat-stress at 35 degrees C (P<0.01). CONCLUSION: EF can retard the aging of C. elegans without damage on the reproductive capacity, and significantly improve its capacity against acute heat-stress.