Tian, Y.; Zhou, Q.; Yu, Q.; Huang, Y.; Zhang, W.; Huang, J.; Yu, T.; Zeng, F.

Ribotoxins disable ribosomes by cleaving ribosomal RNA, but how repair enzymes recognize and restore these damaged machines remains unclear. Here, we report three cryo-electron microscopy structures of the RNA ligase RtcB2 bound to damaged Escherichia coli 30S subunits. An extended interface stabilizes the ribosomal head and decoding-center scaffold while positioning the cleaved 3'-phosphate end within the catalytic pocket. Two structures contain enzyme-linked guanosine monophosphate, whereas the third captures its transfer to the RNA. RNA activation accompanies RtcB2 reorientation and entry of the 5'-hydroxyl-containing segment into an adjacent cleft. Comparisons with assembled ribosomes reveal structural barriers to substrate access. These structures connect recognition of a damaged ribosome to chemical activation of its RNA for repair.