The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3′-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

Targeting the RNA genome of SARS‐CoV‐2 is a viable option for antiviral drug development. We explored three ligand binding sites of the core pseudoknot RNA of the SARS‐CoV‐2 frameshift element. We iteratively optimized ligands, based on improved affinities, targeting these binding sites and report on structural and dynamic properties of the three identified binding sites. Available experimental 3D structures of the pseudoknot element were compared to SAXS and NMR data to validate its dominant folding state in solution. In order to experimentally map in silico predicted binding sites, NMR assignments of the majority of nucleobases were achieved by segmental labeling of the pseudoknot RNA and isotope‐filtered NMR experiments at 1.2 GHz, demonstrating the value of NMR spectroscopy to supplement modelling and docking data. Optimized ligands with enhanced affinity were shown to specifically inhibit frameshifting without affecting 0‐frame translation in cell‐free translation assays, establishing the frameshift element as target for drug‐like ligands of low molecular weight.

Targeting the RNA genome of SARS‐CoV‐2 is a viable option for antiviral drug development. We explored three ligand binding sites of the core pseudoknot RNA of the SARS‐CoV‐2 frameshift element. We iteratively optimized ligands, based on improved affinities, targeting these binding sites and report on structural and dynamic properties of the three identified binding sites. Available experimental 3D structures of the pseudoknot element were compared to SAXS and NMR data to validate its dominant folding state in solution. In order to experimentally map in silico predicted binding sites, NMR assignments of the majority of nucleobases were achieved by segmental labeling of the pseudoknot RNA and isotope‐filtered NMR experiments at 1.2 GHz, demonstrating the value of NMR spectroscopy to supplement modelling and docking data. Optimized ligands with enhanced affinity were shown to specifically inhibit frameshifting without affecting 0‐frame translation in cell‐free translation assays, establishing the frameshift element as target for drug‐like ligands of low molecular weight.

Several SARS-CoV-2 variants have evolved with clinical relevance due to their structural and functional impact on viral proteins and genomic RNA structures. A comprehensive structural and functional characterization of single-nucleotide variations in the 5′-untranslated region (UTR) of SARS-CoV-2 has remained elusive. The co-evolution of 5′-UTR stem-loop 1 (SL1) and non-structural protein (nsp) 1 mutants are of particular importance, as both are key in directing the translation of the viral genome. Here, we investigate the structure and function of the most frequently emerging mutations in SL1 and nsp1. Mutation C21U in the apical loop of SL1 shows changed loop dynamics and reduced escape from repression by nsp1 mutants. Mutation analyses of the pyrimidine loop and the apical helix of SL1 identify preferred sequence motifs for escape from nsp1 repression. Importantly, sequence preferences are governed by the structural features, with suboptimal pyrimidine sequences escaping repression when presented in the SL1 context. Compared to the nsp1 wild type (wt), the currently circulating nsp1 linker variant S135R is much more sensitive toward sequence and structure variations of the apical loop of SL1. Thus, our study provides new insights into the structure–function relationship and co-evolution between viral RNA structures and viral proteins.

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that – when inserted into the human enzyme - confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3’-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.