The LudP arrest peptide stalls translation by remodeling the ribosomal A-site

Bacterial ribosome arrest peptides couple translation to secretion and membrane homeostasis, providing an intracellular regulatory feedback system. Although several Sec/YidC-associated arrest peptides have been characterized, how evolutionarily diverse peptides converge on equivalent physiological outcomes remains unclear. Here we identify LudP (Lacrimispora upstream of secDF peptide), a previously unrecognized arrest peptide conserved in the genus Lacrimispora and encoded immediately upstream of secDF. In vitro and in vivo assays show that LudP induces robust Gram-positive-specific translation arrest that is released in a Sec signal-dependent manner. Using systematic mutagenesis and deep mutational scanning, we define a compact 15-residue C-terminal arrest element that governs arrest stability and A-site residue selectivity. Cryo-electron microscopy reveals that a LudP nascent-chain α-helix in the exit tunnel remodels the peptidyltransferase center, with Asp133-dependent conformational rearrangements of 23S rRNA, notably U2506, sterically and energetically destabilizing productive A-site aminoacyl-tRNA accommodation. These findings support functional convergent evolution of secretion-coupled arrest peptides across bacterial lineages.