A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

Abstract

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-tRNASer 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.

The complete mitochondrial genome of the endemic and highly specialized South African bee speciesRediviva intermixta(Hymenoptera: Melittidae), with a comparison with other bee mitogenomes

“…However, as we used the same prediction method across all taxa, we consider our results comparable. Another structural abnormality found in Rediviva is a lack of the D‐arm of tRNA S1 , but which is similar to almost all metazoan mitogenomes known so far, including other studied Anthophila (Wolstenholme, ; Jühling et al ., ; Suematsu et al ., ). It has been suggested that such an alteration does not affect the function of the tRNA and the molecule is still able to perform aminoacylation and binding to the ribosome (Ohtsuki, Kawai & Watanabe, ; Chimnaronk et al ., ).…”

Analysis of the functional consequences of lethal mutations in mitochondrial translational elongation factors

“…The ability of EF-Tu mt and EF-Tu mt R336Q to protect aa-tRNA from spontaneous deacylation was monitored as described [21], with slight modifications. Ternary complexes were formed in reaction mixtures (100 μL) containing 75 mM Tris-HCl (pH 7.5), 75 mM NH 4 Cl, 15 mM MgCl 2 , 7.5 mM DTT, 60 μg/ml bovine serum albumin, 1 mM GTP, 2.4 mM PEP, 0.1 U pyruvate kinase, 12.5 pmol (0.125 μM) E. coli [ 14 C]Phe-tRNA, and 0–330 pmol (0–3.3 μM) EF-Tu mt (WT or R336Q).…”

Paternal leakage of mitochondrial DNA in experimental crosses of populations of the potato cyst nematode Globodera pallida

“…Purified products were cloned and sequenced as described above, except that either the GeneJET TM Plasmid Miniprep Kit (Fermentas) or the Wizard Ò Plus SV Minipreps DNA Purification System (Promega) were used for plasmid purification. Sequences were analyzed with tRNAscan-SE 1.21 (Lowe and Eddy 1997) to identify tRNA genes, specifying the following: Source, Nematode Mito; Genetic Code for tRNA Isotype Prediction, Invertebrate Mito; and Cove Score Cutoff, 11, due to the observed structural plasticity of nematode tRNA genes (Suematsu et al 2005). …”