Structural basis of ribosomal RNA repair by RtcB2

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.