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Structures of eukaryotic ribonuclease P Ribonuclease P (RNase P) is a ribozyme that processes transfer RNA (tRNA) precursors and is found in all three kingdoms of life. Now, Lan et al. report the structures of yeast RNase P (see the Perspective by Scott and Nagai). The aporibozyme structure reveals how the protein components stabilize the RNA and explains how the structural roles of bacterial RNA elements have been delegated to the protein components in RNase P of higher organisms during evolution. The structure of yeast RNase P in complex with its natural substrate, a tRNA precursor, demonstrates the structural basis for substrate recognition and provides insights into its catalytic mechanism. Science, this issue p. eaat6678; see also p. 644 The structures of ribonuclease P with and without its natural substrate elucidate substrate recognition and catalytic function. INTRODUCTION Ribonuclease P (RNase P), a universal ribozyme that has been found in organisms from all three domains of life, processes the 5′ end of transfer RNA (tRNA). RNase P is a ribonucleoprotein complex, composed of a single catalytic RNA component and a variable number of proteins. Unlike bacterial RNase P, which contains only one small protein cofactor, archaeal and eukaryotic nuclear RNase Ps have evolved considerably more complex protein subunits: five in archaea and 9 to 10 in eukarya. The pre-tRNA processing reaction can be described by a kinetic mechanism that includes four distinct events: (i) rapid and irreversible binding of RNase P (E) to pre-tRNA (S) to form the initial RNase P-pre-tRNA complex (ES); (ii) a conformational change isomerizing the ES complex to a catalytically competent conformer (ES*) in a magnesium ion (Mg2+)–dependent manner; (iii) the cleavage of the phosphodiester bond; and (iv) rapid dissociation of the 5′ leader and slow, rate-limiting release of the mature tRNA (see the figure, right). RATIONALE Despite extensive biochemical and genetic studies, however, the role of protein components and the reason for the increased complexity of the protein moieties in eukaryotic nuclear RNase P are still poorly understood. It is still enigmatic how the pre-tRNA substrate, especially the 5′-leader, is recognized by eukaryotic RNase P; how the catalytically important metal ions are coordinated in the active site; and what the chemical mechanism is of pre-tRNA 5′ cleavage. High-resolution structures of eukaryotic RNase Ps are required to answer these key questions. RESULTS Here, we report the 3.5-Å cryo–electron microscopy structures of Saccharomyces cerevisiae RNase P holoenzyme alone and in complex with pre-tRNAPhe. The yeast RNase P holoenzyme consists of one catalytic RNA Rpr1 and nine protein components. The Rpr1 RNA adopts an extended single-layered conformation that maintains a central helical core but lacks most of the long-range RNA-RNA interactions that are essential for structural stability in bacterial RNase P. The protein components form an interconnected hook-shaped architecture that tightly wraps around the RNA and stabilizes yeast RNase P into a “measuring device,” with two fixed anchors that recognize the L-shaped structure rather than specific sequences of pre-tRNA substrates (see the figure, left). This “measuring device” mediates the initial engagement with pre-tRNA to form the low-affinity ES complex. The recognition of the 5′-leader of pre-tRNA involves both the Rpr1 RNA and the protein subunit Pop5. Two catalytically important Mg2+ ions are coordinated in the catalytic center by highly conserved uridine U93 and the phosphate backbone of Rpr1, together with the scissile phosphate and the O3′ leaving group of pre-tRNA (see the figure, right). The configuration of this RNA-based catalytic center is universally conserved in all RNase Ps, from bacteria to eukarya. Pre-tRNA binding induces a dramatic conformational change in the catalytic center, corresponding to the isomerization step to the ES* state. Moreover, our simulation analysis visualized the mechanistic details of phosphodiester bond hydrolysis of pre-tRNA, which is a two-Mg2+-ion–mediated SN2 reaction (see the figure, right). CONCLUSION The structures presented here represent a major step forward for mechanistic understanding of the function of eukaryotic RNase P. Our data support that all RNase P ribozymes share an RNA-based, substrate-induced catalytic mechanism of pre-RNA processing. Whereas bacterial RNase P RNA is catalytically active by itself, eukaryotic RNase P is a protein-controlled ribozyme; its protein components not only directly participate in substrate recognition but also stabilize the catalytic RNA in a conformation optimal for pre-tRNA binding and cleavage reaction. Catalytic mechanism of pre-tRNA processing catalyzed by yeast RNase P. (Left) The overall structure of yeast RNase P holoenzyme in complex with pre-tRNAPhe. The protein hook and the RNAs [the Rpr1 RNA (gray) and the pre-tRNA (cyan)] are in surface and ribbon representations, respectively. (Right) Pre-tRNA is cleaved by yeast RNase P by means of a kinetic mechanism that includes four distinct events. First, pre-tRNA is recognized by RNase P through a double-anchor mechanism to form the initial ES complex, which induces a local conformational change in the catalytic center of RNase P. In particular, nucleotide U93 of the Rpr1 RNA undergoes a dramatic conformational change to mediate an inner-sphere coordination of the catalytically important Mg2+ ion, so that the ES complex is isomerized to the active ES* state. Next, the activated ES* complex catalyzes the phosphodiester bond cleavage of pre-tRNA through a two-metal-ion SN2 mechanism to release the 5′-leader of pre-tRNA. Last, the mature tRNA dissociates from the holoenzyme in a slow, rate-limiting step, and RNase P is ready for the next round of catalysis. Ribonuclease P (RNase P) is a universal ribozyme responsible for processing the 5′-leader of pre–transfer RNA (pre-tRNA). Here, we report the 3.5-angstrom cryo–electron microscopy structures of Saccharomyces cerevisiae RNase P alone and in complex with pre-tRNAPhe. The protein components form a hook-shaped architecture that wraps around the RNA and stabilizes RNase P into a “measuring device” with two fixed anchors that recognize the L-shaped pre-tRNA. A universally conserved uridine nucleobase and phosphate backbone in the catalytic center together with the scissile phosphate and the O3′ leaving group of pre-tRNA jointly coordinate two catalytic magnesium ions. Binding of pre-tRNA induces a conformational change in the catalytic center that is required for catalysis. Moreover, simulation analysis suggests a two-metal-ion SN2 reaction pathway of pre-tRNA cleavage. These results not only reveal the architecture of yeast RNase P but also provide a molecular basis of how the 5′-leader of pre-tRNA is processed by eukaryotic RNase P.