Three-metal-ion catalysis by ribonuclease P holoenzyme: a new mechanistic insight into transfer RNA maturation

Ribonuclease P (RNase P) is an essential metalloenzyme responsible for tRNA 5′ maturation across all three domains of life. The catalytic mechanism, particularly the number of divalent metal ions and the role of its protein component, remains unresolved. We determined cryo-EM structures of Geobacillus Stearothermophilus RNase P holoenzyme at 2.7-3.0 Å resolution in three catalytic states. These structures reveal a three-metal-ion catalytic mechanism, challenging the long-standing two-metal-ion model. Two metal ions are substrate-dependent, one of which is also protein-dependent. The protein component, rnpA, dramatically enhances ribozyme activity by reducing conformational sampling, stabilizing the enzyme-substrate complex through direct contact with the pre-tRNA 5′-leader, and cooperatively stabilizing the third catalytic metal ion with the substrate.