Structural Basis for Translational Stalling by Human Cytomegalovirus and Fungal Arginine Attenuator Peptide

Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes. [Display omitted] ► Preparation of in vivo-stalled eukaryotic ribosome-nascent chain complexes ► The arginine attenuator peptide is compacted within the tunnel adjacent to the PTC ► Nascent chains of AAP- and CMV-stalled RNCs are stabilized at the constriction ► Eukaryotic-specific loop of L10e directly contacts the CCA end of the P-tRNA