Mimicry of Canonical Translation Elongation Underlies Alanine Tail Synthesis in RQC

Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation. [Display omitted] •Structural basis for nascent chain modification with Ala tails in bacterial RQC•Specific recruitment of tRNAAla to A-site by RqcH-mediated tRNA anticodon reading•RqcH-mediated formation of an A/P-tRNA state associated with peptidyl transfer•Hsp15/RqcP completes the cycle by stabilizing the P-site tRNA conformation Incompletely made nascent chains are tagged for proteolysis by ribosome-associated quality control (RQC). In bacterial RQC, this is mediated by C-terminal alanine tailing. Filbeck et al. elucidate the structural basis for alanine tailing using cryo-EM, discover Hsp15/RqcP as an essential factor, and reveal that C-terminal tailing follows similar principles to canonical translation elongation.