Transfer RNA (tRNA) is subject to a multitude of posttranscriptional modifications which can profoundly impact its functionality as the essential adaptor molecule in messenger RNA (mRNA) translation. Therefore, dynamic regulation of tRNA modification in response to environmental changes can tune the efficiency of gene expression in concert with the emerging epitranscriptomic mRNA regulators. Several of the tRNA modifications are required to prevent human diseases and are particularly important for proper development and generation of neurons. In addition to the positive role of different tRNA modifications in prevention of neurodegeneration, certain cancer types upregulate tRNA modification genes to sustain cancer cell gene expression and metastasis. Multiple associations of defects in genes encoding subunits of the tRNA modifier complex Elongator with human disease highlight the importance of proper anticodon wobble uridine modifications (xm⁵U₃₄) for health. Elongator functionality requires communication with accessory proteins and dynamic phosphorylation, providing regulatory control of its function. Here, we summarized recent insights into molecular functions of the complex and the role of Elongator dependent tRNA modification in human disease.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin‐related modifier 1 (Urm1) pathway is responsible for the synthesis of 2‐thiolated wobble uridine (U ₃₄ ). During the key step of the modification cascade, the E1‐like activating enzyme ubiquitin‐like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C‐terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1‐COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin‐like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin‐like proteins (UBL) and sulfur‐relay systems. Here, we report the crystal structures of full‐length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C‐terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self‐conjugation with its own product, namely activated Urm1‐COSH.

The yeast peroxiredoxin Ahp1, like related anti-oxidant enzymes in other species, undergoes urmylation, a lysine-directed conjugation to ubiquitin-like modifier Urm1. Ahp1 assembles into a homodimer that detoxifies peroxides via forming intersubunit disulfides between peroxidatic and resolving cysteines that are subsequently reduced by the thioredoxin system. Although urmylation coincides with oxidative stress, it is unclear how this modification happens on a molecular level and whether it affects peroxiredoxin activity. Here, we report that thioredoxin mutants decrease Ahp1 urmylation in yeast and each subunit of the oxidized Ahp1 dimer is modified by Urm1 suggesting coupling of urmylation to dimerization. Consistently, Ahp1 mutants unable to form dimers, fail to be urmylated as do mutants that lack the peroxidatic cysteine. Moreover, Ahp1 urmylation involves at least two lysine residues close to the catalytic cysteines and can be prevented in yeast cells exposed to high organic peroxide concentrations. Our results elucidate redox requirements and molecular determinants critical for Ahp1 urmylation, thus providing insights into a potential link between oxidant defense and Urm1 utilization in cells.

Post‐translational modifications by ubiquitin‐like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin‐related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin‐like protein activator 4). While Urm1 has also been observed to conjugate to target proteins like other UBLs, the molecular mechanism of its attachment remains unknown. Here, we reconstitute the covalent attachment of thiocarboxylated Urm1 to various cellular target proteins in vitro, revealing that, unlike other known UBLs, this process is E2/E3‐independent and requires oxidative stress. Furthermore, we present the crystal structures of the peroxiredoxin Ahp1 before and after the covalent attachment of Urm1. Surprisingly, we show that urmylation is accompanied by the transfer of sulfur to cysteine residues in the target proteins, also known as cysteine persulfidation. Our results illustrate the role of the Uba4‐Urm1 system as a key evolutionary link between prokaryotic SCPs and the UBL modifications observed in modern eukaryotes.

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid. A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress. This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.

Industrial production of bioethanol from lignocellulosic materials (LCM′s) is reliant on a microorganism being tolerant to the stresses inherent to fermentation. Previous work has highlighted the importance of a cytochrome oxidase chaperone gene (COX20) in improving yeast tolerance to acetic acid, a common inhibitory compound produced during pre-treatment of LCM’s. The presence of acetic acid has been shown to induce oxidative stress and programmed cell death, so the role of COX20 in oxidative stress was determined. Analysis using flow cytometry revealed that COX20 expression was associated with reduced levels of reactive oxygen species (ROS) in hydrogen peroxide and metal-induced stress, and there was a reduction in apoptotic and necrotic cells when compared with a strain without COX20. Results on the functionality of COX20 have revealed that overexpression of COX20 induced respiratory growth in Δimp1 and Δcox18, two genes whose presence is essential for yeast respiratory growth. COX20 also has a role in protecting the yeast cell against programmed cell death.