The crystal structures of the various complexes formed by yeast aspartyl‐tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA‐AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl‐adenylate and/or the aspartyl‐tRNA. A class II‐specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class‐invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site‐directed mutagenesis for their functional implication.

SARS-CoV-2 coronavirus is responsible for the Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into nonstructural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. Despite the presence of NSP1 on the ribosome, viral translation proceeds, however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1 and we show that the evasion to NSP1-inhibition is mediated by the cis-acting RNA hairpin SL1 in the 5′UTR of SARS-CoV-2. Only the apical part of SL1 is required for viral translation. We further show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation and fostering SARS-CoV-2 translation in the presence of the SL1 5′UTR hairpin. SL1 is also present and necessary for translation of subgenomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine “Achilles heel” of the virus.

The SARS-CoV-2, a positive-sense single-stranded RNA Coronavirus, is a global threat to human health. Thus, understanding its life cycle mechanistically would be important to facilitate the design of antiviral drugs. A key aspect of viral progression is the synthesis of viral proteins by the ribosome of the human host. In Coronaviruses, this process is regulated by the viral 5ʹ and 3ʹ untranslated regions (UTRs), but the precise regulatory mechanism has not yet been well understood. In particular, the 5ʹ-UTR of the viral genome is most likely involved in translation initiation of viral proteins. Here, we performed inline probing and RNase V1 probing to establish a model of the secondary structure of SARS-CoV-2 5ʹ-UTR. We found that the 5ʹ-UTR contains stable structures including a very stable four-way junction close to the AUG start codon. Sequence alignment analysis of SARS-CoV-2 variants 5ʹ-UTRs revealed a highly conserved structure with few co-variations that confirmed our secondary structure model based on probing experiments.

Expansion of G₄C₂ repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded transcripts undergo repeat-associated non-AUG (RAN) translation producing dipeptide repeat proteins from all reading frames. We determined cis-factors and trans-factors influencing translation of the human C9ORF72 transcripts. G₄C₂ translation operates through a 5′–3′ cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNA^Met_i. Production of poly-GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG start codon supporting a frameshifting mechanism. RAN translation is also regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5′-flanking G₄C₂ sequence block ribosomal scanning and prevent translation. Finally, we identified an unexpected affinity of expanded transcripts for the ribosomal subunits independently from translation.