The eukaryote-specific N-terminal domain of ribosomal protein eS31 is a flexible extension near the decoding center, but its role in translational control has been obscured by pleiotropic defects associated with complete domain deletion. To resolve this ambiguity, we dissected its function at amino acid resolution in Saccharomyces cerevisiae using a genetic screen for ribosome stalling at CGA codon repeats. By functionally separating the N-terminal ubiquitin moiety from the ribosomal domain, our screen bypassed artifacts and identified a critical cluster of basic residues (K79-K82) within this region. Charge-reversing point mutations in this cluster alleviate stalling not by impairing ribosome-associated quality control, but by reducing decoding fidelity, as supported by paromomycin sensitivity and genetic suppression by the loss of the eEF2 diphthamide modification. Crucially, the mutant lacking the entire domain operates through a fundamentally distinct mechanism, exhibiting a severe defect in termination fidelity and a paradoxical requirement for wild-type eS31 that suggests a global structural perturbation. Our findings thus resolve the function of the N-terminal domain of eS31 into two distinct modes: a residue-specific, electrostatic role in fine-tuning elongation fidelity, and a broader structural role for the entire domain in maintaining ribosome integrity for accurate termination.
Gao et al. (Tue,) studied this question.