Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation.

TRNAs are key partners in ribosome-dependent protein synthesis. This process is highly dependent on the fidelity of tRNA aminoacylation by aminoacyl-tRNA synthetases and relies primarily on sets of identities within tRNA molecules composed of determinants and antideterminants preventing mischarging by non-cognate synthetases. Such identity sets were discovered in the tRNAs of a few model organisms, and their properties were generalized as universal identity rules. Since then, the panel of identity elements governing the accuracy of tRNA aminoacylation has expanded considerably, but the increasing number of reported functional idiosyncrasies has led to some confusion. In parallel, the description of other processes involving tRNAs, often well beyond aminoacylation, has progressed considerably, greatly expanding their interactome and uncovering multiple novel identities on the same tRNA molecule. This review highlights key findings on the mechanistics and evolution of tRNA and tRNA-like identities. In addition, new methods and their results for searching sets of multiple identities on a single tRNA are discussed. Taken together, this knowledge shows that a comprehensive understanding of the functional role of individual and collective nucleotide identity sets in tRNA molecules is needed for medical, biotechnological and other applications.

Aminoacyl-tRNA synthetases (aaRSs) are modular enzymes globally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e., the attachment of a proteinogenic amino acid on their cognate tRNAs, thereby mediating the correct expression of the genetic code. In addition, some aaRSs acquired other functions beyond this key role in translation. Genomics and X-ray crystallography have revealed great structural diversity in aaRSs (e.g., in oligomery and modularity, in ranking into two distinct groups each subdivided in 3 subgroups, by additional domains appended on the catalytic modules). AaRSs show huge structural plasticity related to function and limited idiosyncrasies that are kingdom or even species specific (e.g., the presence in many Bacteria of non discriminating aaRSs compensating for the absence of one or two specific aaRSs, notably AsnRS and/or GlnRS). Diversity, as well, occurs in the mechanisms of aaRS gene regulation that are not conserved in evolution, notably between distant groups such as Gram-positive and Gram-negative Bacteria . The review focuses on bacterial aaRSs (and their paralogs) and covers their structure, function, regulation, and evolution. Structure/function relationships are emphasized, notably the enzymology of tRNA aminoacylation and the editing mechanisms for correction of activation and charging errors. The huge amount of genomic and structural data that accumulated in last two decades is reviewed, showing how the field moved from essentially reductionist biology towards more global and integrated approaches. Likewise, the alternative functions of aaRSs and those of aaRS paralogs (e.g., during cell wall biogenesis and other metabolic processes in or outside protein synthesis) are reviewed. Since aaRS phylogenies present promiscuous bacterial, archaeal, and eukaryal features, similarities and differences in the properties of aaRSs from the three kingdoms of life are pinpointed throughout the review and distinctive characteristics of bacterium-like synthetases from organelles are outlined.

The evolution of biocrystallography from the pioneers' time to the present era of global biology is presented in relation to the development of methodological and instrumental advances for molecular sample preparation and structure elucidation over the last 6 decades. The interdisciplinarity of the field that generated cross‐fertilization between physics‐ and biology‐focused themes is emphasized. In particular, strategies to circumvent the main bottlenecks of biocrystallography are discussed. They concern (i) the way macromolecular targets are selected, designed, and characterized, (ii) crystallogenesis and how to deal with physical and biological parameters that impact crystallization for growing and optimizing crystals, and (iii) the methods for crystal analysis and 3D structure determination. Milestones that have marked the history of biocrystallography illustrate the discussion. Finally, the future of the field is envisaged. Wide gaps of the structural space need to be filed and membrane proteins as well as intrinsically unstructured proteins still constitute challenging targets. Solving supramolecular assemblies of increasing complexity, developing a “4D biology” for decrypting the kinematic changes in macromolecular structures in action, integrating these structural data in the whole cell organization, and deciphering biomedical implications will represent the new frontiers.

The solution conformation of eight leucine tRNAs from Phaseolus vulgaris, baker's yeast and Escherichia coli, characterized by long variable regions, and the interaction of four of them with bean cytoplasmic leucyl-tRNA synthetase were studied by phosphate mapping with ethylnitrosourea. Phosphate reactivities in the variable regions agree with the existence of RNA helices closed by miniloops. At the junction of these regions with the T-stem, phosphate 48 is strongly protected, in contrast to small variable region tRNAs where P49 is protected. The constant protection of P22 is another characteristics of leucine tRNAs. Conformational differences between leucine isoacceptors concern the anticodon region, the D-arm and the variable region. In several parts of free tRNALeu species, e.g. in the T-loop, phosphate reactivities are similar to those found in tRNAs of other specificities, indicating conformational similarities among tRNAs. Phosphate alkylation of four leucine tRNAs complexed to leucyl-tRNA synthetase indicates that the 3'-side of the anticodon stem, the D-stem and the hinge region between the anticodon and D-stems are in contact with the plant enzyme.