Switching a conflicted bacterial DTD-tRNA code is essential for the emergence of mitochondria.

Mitochondria emerged through an endosymbiotic event involving a proteobacterium and an archaeal host. However, the process of optimization of cellular processes required for the successful evolution and survival of mitochondria, which integrates components from two evolutionarily distinct ancestors as well as novel eukaryotic elements, is not well understood. We identify two key switches in the translational machinery­one in the discriminator recognition code of a chiral proofreader DTD [d-aminoacyl­transfer RNA (tRNA) deacylase] and the other in mitochondrial tRNAGly­that enable the compatibility between disparate elements essential for survival. Notably, the mito-tRNAGly discriminator element is the only one to switch from pyrimidine to purine during the bacteria-to-mitochondria transition. We capture this code transition in the Jakobida, an early diverging eukaryotic clade bearing the most bacterial-like mito-genome, wherein both discriminator elements are present. This study underscores the need to explore the fundamental integration strategies critical for mitochondrial and eukaryotic evolution.

Genomic innovation of ATD alleviates mistranslation associated with multicellularity in Animalia.

The emergence of multicellularity in Animalia is associated with increase in ROS and expansion of tRNA-isodecoders. tRNA expansion leads to misselection resulting in a critical error of L-Ala mischarged onto tRNAThr, which is proofread by Animalia-specific-tRNA Deacylase (ATD) in vitro. Here we show that in addition to ATD, threonyl-tRNA synthetase (ThrRS) can clear the error in cellular scenario. This two-tier functional redundancy for translation quality control breaks down during oxidative stress, wherein ThrRS is rendered inactive. Therefore, ATD knockout cells display pronounced sensitivity through increased mistranslation of threonine codons leading to cell death. Strikingly, we identify the emergence of ATD along with the error inducing tRNA species starting from Choanoflagellates thus uncovering an important genomic innovation required for multicellularity that occurred in unicellular ancestors of animals. The study further provides a plausible regulatory mechanism wherein the cellular fate of tRNAs can be switched from protein biosynthesis to non-canonical functions.

The first animals evolved around 750 million years ago from single-celled ancestors that were most similar to modern-day organisms called the Choanoflagellates. As animals evolved they developed more complex body plans consisting of multiple cells organized into larger structures known as tissues and organs. Over time cells also evolved increased levels of molecules called reactive oxygen species, which are involved in many essential cell processes but are toxic at high levels. Animal cells also contain more types of molecules known as transfer ribonucleic acids, or tRNAs for short, than Choanoflagellate cells and other single-celled organisms. These molecules deliver building blocks known as amino acids to the machinery that produces new proteins. To ensure the proteins are made correctly, it is important that tRNAs deliver specific amino acids to the protein-building machinery in the right order. Each type of tRNA usually only pairs with a specific type of amino acid, but sometimes the enzymes involved in this process can make mistakes. Therefore, cells contain proofreading enzymes that help remove incorrect amino acids on tRNAs. One such enzyme ­ called ATD ­ is only found in animals. Experiments in test tubes reported that ATD removes an amino acid called alanine from tRNAs that are supposed to carry threonine, but its precise role in living cells remained unclear. To address this question, Kuncha et al. studied proofreading enzymes in human kidney cells. The experiments showed that, in addition to ATD, a second enzyme known as ThrRS was also able to correct alanine substitutions for threonines on tRNAs. However, reactive oxygen species inactivated the proofreading ability of ThrRS, suggesting ATD plays an essential role in correcting errors in cells containing high levels of reactive oxygen species. These findings suggest that as organisms evolved multiple cells and the levels of tRNA and oxidative stress increased, this led to the appearance of a new proofreading enzyme. Further studies found that ATD originated around 900 million years ago, before Choanoflagellates and animals diverged, indicating these enzymes might have helped to shape the evolution of animals. The next step following on from this work will be to understand the role of ATD in the cells of organs that are known to have particularly high levels of reactive oxygen species, such as testis and ovaries.

A discriminator code-based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria.

D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, attached to tRNA. We have recently shown that this activity enables DTD to clear non-cognate Gly-tRNAAla with 1000-fold higher efficiency than its activity on Gly-tRNAGly, indicating tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's discriminator base predominantly accounts for this activity difference and is the key to selection by DTD. Accordingly, the uracil discriminator base, serving as a negative determinant, prevents Gly-tRNAGly misediting by DTD and this protection is augmented by EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only G3•U70 for tRNAGly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine misincorporation in proteins rather than only recycling mischarged tRNAAla. Overall, the study reveals the unique co-evolution of DTD and discriminator base, and suggests DTD's strong selection pressure on bacterial tRNAGlys to retain a pyrimidine discriminator code.

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of a female-specific lipocalin (FLP) expressed in the lacrimal glands of Syrian hamsters.

Proteins belonging to the lipocalin superfamily are usually secretory proteins of molecular mass approximately 20 kDa with a hydrophobic pocket for the binding and transport of diverse small ligands. Various lipocalins have been associated with many biological processes, e.g. immunomodulation, odorant transport, pheromonal activity, retinoid transport, cancer-cell interactions etc. However, the exact functions of many lipocalins and the ligands bound by them are unclear. Previously, the cDNA of a 20 kDa lipocalin (FLP) which is female-specifically expressed in the lacrimal glands of Syrian (golden) hamsters and secreted in the tears of females has been identified and cloned. His-tagged recombinant FLP (rFLP) has now been cloned, overexpressed in Escherichia coli as a soluble protein and purified to homogeneity using Ni-affinity followed by size-exclusion chromatography. Purified rFLP was crystallized using the sitting-drop vapour-diffusion method. The crystals tested belonged to space group P2(1)2(1)2(1) and diffracted to beyond 1.86 A resolution. Solvent-content analysis indicated the presence of one monomer in the asymmetric unit.