Structural basis of transfer RNA processing by bacterial minimal RNase P

Precursor tRNAs (pre-tRNAs) require nucleolytic removal of 5'-leader and 3'-trailer sequences for maturation, which is essential for proper tRNA function. The endoribonuclease RNase P exists in diverse forms, including RNA- and protein-based RNase P, and removes 5'-leader sequences from pre-tRNAs. Some bacteria and archaea possess a unique minimal protein-based RNase P enzyme, HARP, which forms dodecamers with twelve active sites. Here, we present cryogenic electron microscopy structures of HARP dodecamers complexed with five pre-tRNAs, and we show that HARP oligomerization enables specific recognition of the invariant distance between the acceptor stem 5'-end and the TψC-loop, functioning as a molecular ruler-a feature representing convergent evolution among RNase P enzymes. The HARP dodecamer uses only five active sites for 5'-leader cleavage, while we identify a 3'-trailer cleavage activity in the remaining seven sites. This elucidation reveals how small proteins evolve through oligomerization to adapt a pivotal biological function (5'-leader processing) and acquire a novel function (3'-trailer processing).