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A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNATyr molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNATyr tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNATyr molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNATyr via nonclassical Watson–Crick G15–C48 and G13–A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNATyr life cycle.

Aminoacyl-tRNA synthetases are pivotal in determining how the genetic code is translated in amino acids and in providing the substrate for protein synthesis. As such, they fulfill a key role in a process universally conserved in all cellular organisms from their most complex to their most reduced parasitic forms. In contrast, even complex viruses were not found to encode much translation machinery, with the exception of isolated components such as tRNAs. In this context, the discovery of four aminoacyl-tRNA synthetases encoded in the genome of mimivirus together with a full set of translation initiation, elongation, and termination factors appeared to blur what was once a clear frontier between the cellular and viral world. Functional studies of two mimivirus tRNA synthetases confirmed the MetRS specificity for methionine and the TyrRS specificity for tyrosine and conformity with the identity rules for tRNA Tyr for archea/eukarya. The atomic structure of the mimivirus tyrosyl-tRNA synthetase in complex with tyrosinol exhibits the typical fold and active-site organization of archaeal-type TyrRS. However, the viral enzyme presents a unique dimeric conformation and significant differences in its anticodon binding site. The present work suggests that mimivirus aminoacyl-tRNA synthetases function as regular translation enzymes in infected amoebas. Their phylogenetic classification does not suggest that they have been acquired recently by horizontal gene transfer from a cellular host but rather militates in favor of an intricate evolutionary relationship between large DNA viruses and ancestral eukaryotes.

Most bacteria and eukarya contain an enzyme capable of specifically hydrolyzing D-aminoacyl-tRNA. Here, the archaea Sulfolobus solfataricus is shown to also contain an enzyme activity capable of recycling misaminoacylated D-Tyr-tRNATyr. N-terminal sequencing of this enzyme identifies open reading frame SS02234 (dtd2), the product of which does not present any sequence homology with the known D-Tyr-tRNATyr deacylases of bacteria or eukaryotes. On the other hand, homologs of dtd2 occur in archaea and plants. The Pyrococcus abyssi dtd2 ortholog (PAB2349) was isolated. It rescues the sensitivity to D-tyrosine of a mutant Escherichia coli strain lacking dtd, the gene of its endogeneous D-Tyr-tRNATyr deacylase. Moreover, in vitro, the PAB2349 product, which behaves as a monomer and carries 2 mol of zinc/mol of protein, catalyzes the cleavage of D-Tyr-tRNATyr. The three-dimensional structure of the product of the Archaeoglobus fulgidus dtd2 ortholog has been recently solved by others through a structural genomics approach (Protein Data Bank code 1YQE). This structure does not resemble that of Escherichia coli D-Tyr-tRNATyr deacylase. Instead, it displays homology with that of a bacterial peptidyl-tRNA hydrolase. We show, however, that the archaeal PAB2349 enzyme does not act against diacetyl-Lys-tRNALys, a model substrate of peptidyl-tRNA hydrolase. Based on the Protein Data Bank 1YQE structure, site-directed mutagenesis experiments were undertaken to remove zinc from the PAB2349 enzyme. Several residues involved in zinc binding and supporting the activity of the deacylase were identified. Taken together, these observations suggest evolutionary links between the various hydrolases in charge of the recycling of metabolically inactive tRNAs during translation.

International audience; The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes.

International audience; The Saccharomyces cerevisiae YDL219w (DTD1) gene, which codes for an amino acid sequence sharing 34% identity with the Escherichia coli D-Tyr-tRNA(Tyr) deacylase, was cloned, and its product was functionally characterized. Overexpression in the yeast of the DTD1 gene from a multicopy plasmid increased D-Tyr-tRNA(Tyr) deacylase activity in crude extracts by two orders of magnitude. Upon disruption of the chromosomal gene, deacylase activity was decreased by more than 90%, and the sensitivity to D-tyrosine of the growth of S. cerevisiae was exacerbated. The toxicity of D-tyrosine was also enhanced under conditions of nitrogen starvation, which stimulate the uptake of D-amino acids. In relation with these behaviors, the capacity of purified S. cerevisiae tyrosyl-tRNA synthetase to produce D-Tyr-tRNA(Tyr) could be shown. Finally, the phylogenetic distribution of genes homologous to DTD1 was examined in connection with L-tyrosine prototrophy or auxotrophy. In the auxotrophs, DTD1-like genes are systematically absent. In the prototrophs, the putative occurrence of a deacylase is variable. It possibly depends on the L-tyrosine anabolic pathway adopted by the cell.

International audience; FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.