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Free keywords:
Carbon-Nitrogen Ligases with Glutamine as; Amide-N-Donor/biosynthesis/chemistry/genetics; Circular Dichroism; Cryoelectron Microscopy; Cytomegalovirus/genetics/metabolism; Gene Expression Regulation, Fungal; Magnetic Resonance Spectroscopy; Models, Molecular; Nucleic Acid Conformation; Open Reading Frames; Peptide Fragments/biosynthesis/chemistry/genetics; Peptidyl Transferases/chemistry; Protein Biosynthesis; Protein Conformation; RNA, Transfer, Amino Acyl/chemistry; Ribosomal Proteins/chemistry; Ribosomes/metabolism/ultrastructure; Structure-Activity Relationship; Viral Envelope Proteins/biosynthesis/chemistry/genetics; Yeasts/genetics/metabolism
Abstract:
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.