RESEARCH ARTICLE SUMMARY◥

STRUCTURAL BIOLOGY

Structural insight into precursortRNA processing by yeastribonuclease PPengfei Lan*, Ming Tan*, Yuebin Zhang*, Shuangshuang Niu, Juan Chen, Shaohua Shi,Shuwan Qiu, Xuejuan Wang, Xiangda Peng, Gang Cai, Hong Cheng, Jian Wu†,Guohui Li†, Ming Lei†

INTRODUCTION: Ribonuclease P (RNase P),a universal ribozyme that has been found inorganisms from all three domains of life, pro-cesses the 5′ end of transfer RNA (tRNA). RNase Pis a ribonucleoprotein complex, composedof a single catalytic RNA component and avariable number of proteins. Unlike bacterialRNase P, which contains only one small pro-tein cofactor, archaeal and eukaryotic nuclearRNase Ps have evolved considerably more com-plex protein subunits: five in archaea and 9 to10 in eukarya. The pre-tRNA processing reac-tion can be described by a kinetic mechanismthat includes four distinct events: (i) rapidand irreversible binding of RNase P (E) to pre-tRNA (S) to form the initial RNase P-pre-tRNAcomplex (ES); (ii) a conformational change

isomerizing the ES complex to a catalyticallycompetent conformer (ES*) in a magnesiumion (Mg2+)–dependent manner; (iii) the cleav-age of the phosphodiester bond; and (iv) rapiddissociation of the 5′ leader and slow, rate-limiting release of the mature tRNA (see thefigure, right).

RATIONALE:Despite extensive biochemicaland genetic studies, however, the role of pro-tein components and the reason for the in-creased complexity of the protein moieties ineukaryotic nuclear RNase P are still poorly un-derstood. It is still enigmatic how the pre-tRNAsubstrate, especially the 5′-leader, is recognizedby eukaryotic RNase P; how the catalyticallyimportant metal ions are coordinated in the

active site; and what the chemical mechanismis of pre-tRNA 5′ cleavage. High-resolution struc-tures of eukaryotic RNase Ps are required toanswer these key questions.

RESULTS: Here, we report the 3.5-Å cryo–electron microscopy structures of Saccharomycescerevisiae RNase P holoenzyme alone and incomplex with pre-tRNAPhe. The yeast RNase Pholoenzyme consists of one catalytic RNA Rpr1

and nine protein compo-nents. TheRpr1RNAadoptsan extendedsingle-layeredconformation that main-tains a central helical corebut lacksmost of the long-range RNA-RNA interac-

tions that are essential for structural stability inbacterial RNase P. The protein components forman interconnected hook-shaped architecture thattightly wraps around the RNA and stabilizesyeast RNase P into a “measuring device,” withtwo fixed anchors that recognize the L-shapedstructure rather than specific sequences of pre-tRNA substrates (see the figure, left). This “mea-suring device”mediates the initial engagementwith pre-tRNA to form the low-affinity ES com-plex. The recognition of the 5′-leader of pre-tRNAinvolves both the Rpr1 RNA and the proteinsubunit Pop5. Two catalytically important Mg2+

ions are coordinated in the catalytic center byhighly conserved uridine U93 and the phos-phate backbone of Rpr1, together with thescissile phosphate and the O3′ leaving group ofpre-tRNA (see the figure, right). The configurationof this RNA-based catalytic center is universallyconserved inallRNasePs, frombacteria toeukarya.Pre-tRNA binding induces a dramatic conforma-tional change in the catalytic center, correspond-ing to the isomerization step to the ES* state.Moreover, our simulation analysis visualized themechanistic details of phosphodiester bond hy-drolysis of pre-tRNA, which is a two-Mg2+-ion–mediated SN2 reaction (see the figure, right).

CONCLUSION: The structures presented hererepresent amajor step forward formechanisticunderstanding of the function of eukaryoticRNase P. Our data support that all RNase Pribozymes share an RNA-based, substrate-induced catalytic mechanism of pre-RNA pro-cessing. Whereas bacterial RNase P RNA iscatalytically active by itself, eukaryotic RNaseP is a protein-controlled ribozyme; its proteincomponents not only directly participate insubstrate recognition but also stabilize thecatalytic RNA in a conformation optimal forpre-tRNA binding and cleavage reaction.▪

RESEARCH

Lan et al., Science 362, 657 (2018) 9 November 2018 1 of 1

The list of author affiliations is available in the full article online.*These authors contributed equally to this work.†Corresponding author. Email: leim@shsmu.edu.cn (M.L.);ghli@dicp.ac.cn (G.L.); wujian@shsmu.edu.cn (J.W.)Cite this article as P. Lan et al., Science 362, eaat6678(2018). DOI: 10.1126/science.aat6678

ProteinsRpr1S-domainRpr1C-domainU93Active centertRNA5’-leader

Arm

Pre-tRNA

ES

5’-leader

ES*EP

Head

Rpr1

P

Mature tRNA

E

SPre-tRNA

Catalytic mechanism of pre-tRNA processing catalyzed by yeast RNase P. (Left) Theoverall structure of yeast RNase P holoenzyme in complex with pre-tRNAPhe. The protein hookand the RNAs [the Rpr1 RNA (gray) and the pre-tRNA (cyan)] are in surface and ribbonrepresentations, respectively. (Right) Pre-tRNA is cleaved by yeast RNase P by means of akinetic mechanism that includes four distinct events. First, pre-tRNA is recognized by RNase Pthrough a double-anchor mechanism to form the initial ES complex, which induces a localconformational change in the catalytic center of RNase P. In particular, nucleotide U93 of theRpr1 RNA undergoes a dramatic conformational change to mediate an inner-spherecoordination of the catalytically important Mg2+ ion, so that the ES complex is isomerized tothe active ES* state. Next, the activated ES* complex catalyzes the phosphodiester bondcleavage 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.

ON OUR WEBSITE◥

Read the full articleat http://dx.doi.org/10.1126/science.aat6678..................................................

on Septem

ber 30, 2020

http://science.sciencemag.org/

Dow

nloaded from

RESEARCH ARTICLE◥

STRUCTURAL BIOLOGY

Structural insight into precursortRNA processing by yeastribonuclease PPengfei Lan1*, Ming Tan2,3*, Yuebin Zhang4*, Shuangshuang Niu2,3,5, Juan Chen1,Shaohua Shi1, Shuwan Qiu6, Xuejuan Wang6, Xiangda Peng4, Gang Cai6, Hong Cheng2,Jian Wu1†, Guohui Li4†, Ming Lei1,7,8,9†

Ribonuclease P (RNase P) is a universal ribozyme responsible for processing the 5′-leaderof pre–transfer RNA (pre-tRNA). Here, we report the 3.5-angstrom cryo–electronmicroscopy structures of Saccharomyces cerevisiae RNase P alone and in complexwith pre-tRNAPhe. The protein components form a hook-shaped architecture that wrapsaround the RNA and stabilizes RNase P into a “measuring device” with two fixedanchors that recognize the L-shaped pre-tRNA. A universally conserved uridine nucleobaseand phosphate backbone in the catalytic center together with the scissile phosphateand 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 isrequired for catalysis. Moreover, simulation analysis suggests a two-metal-ion SN2 reactionpathway of pre-tRNA cleavage. These results not only reveal the architecture of yeastRNase P but also provide a molecular basis of how the 5′-leader of pre-tRNA is processedby eukaryotic RNase P.

Ribonuclease P (RNase P), one of only twouniversal ribozymes that have been foundin organisms from all three domains oflife, is responsible for the maturation ofthe 5′ end of transfer RNA (tRNA) (1).

RNase P is ribonucleoprotein complex, com-posed of a single catalytic RNA component anda variable number of proteins (2–4). The RNAcomponent of RNase P from all organisms showsmarked similarities at the primary and second-ary structure level, which is suggestive of thepresence of a universally conserved catalytic RNAcore (2, 4). All RNase P RNAs can be divided intotwo independent folded domains, the catalyticdomain (C domain) and the specificity domain(S domain), which play key roles in substratecleavage and substrate binding, respectively (5, 6).Bacterial RNase P contains a single small protein(Rpp) that is essential for substrate recognitionand cleavage under physiological conditions (7–9).By contrast, archaeal and eukaryotic nuclearRNase Ps have evolved considerably more com-plex subunit compositions with an increasednumber of protein components, five in archaeaand 9 to 10 in eukarya (2). Unlike bacterial RNAs,

the RNAs themselves in archaeal and eukaryoticRNase Ps are not generally catalytically active,and protein subunits are required to enhancethe pre-tRNA substrate binding affinity andcleavage efficiency (10–13).Very little is known about the structure of eu-

karyotic RNase Ps. Only crystal structures of hu-man Pop6-Pop7 subcomplex and Saccharomycescerevisiae Pop6-Pop7 complexed with the P3element of RNase MRP (mitochondrial RNAprocessing) RNAare available (14, 15). In addition,a low-resolution structure of S. cerevisiae nuclearRNase P has been solved by means of cryo–negative staining electron microscopy (EM) (16).A high-resolution structure of eukaryotic nuclearRNase P holoenzyme still has yet to be de-termined, hindering our understanding of thestructural organization andmechanism of actionof RNase P from higher organisms.RNase P is a multiple turnover ribozyme that

recognizes its substrates in trans (17). Previousstudies suggest that the cleavage of pre-tRNA byRNase P can be described by a kinetic mecha-nism that includes at least 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) the ES complex thenundergoes a conformational change and is isom-erized to a catalytically competent conformer(ES) in a magnesium ion(Mg2+)–dependentmanner; (iii) the cleavage of the phosphodiesterbond; and (iv) rapid dissociation of the 5′-leaderand slow, rate-limiting release of the maturetRNA (18–21). Although this kinetic model wasproposed about a decade ago, how RNase P fa-cilitates such a reaction remains largely enigmatic.The first step of this kinetic scheme represents

the formation of the initial low-affinity ES com-plex. RNase P recognizes the three-dimensionalstructural feature rather than specific sequencesof pre-tRNAs (1, 22, 23). It has been proposedthat RNase P contains a “measuring device” thatrecognizes a common structural feature of allpre-tRNAs; the coaxially stacked acceptor andT stems have a fixed length of 12 base pairs ofnucleotides (1). The crystal structure of the bacte-ria Thermotoga maritima RNase P–tRNA complexwith a soaked 5′-leader provides the structuralbasis of a simple, RNA-based “measuring device”and some insight into the binding of pre-tRNA5′-leader (23). However, pre-tRNA recognition bythe much more complex and indispensable pro-tein components of eukaryotic RNase Ps is stillpoorly understood. Kinetic evidences suggestedthat a conformational change occurs after theformation of the initial ES complex and before5′-leader cleavage, transforming the ES complexinto an active ES* state (7, 9, 18, 24, 25). The ES*complex is stabilized by at least two Mg2+ ionsduring the transition from ES to ES*, and thefunctional groups and metal ions in the activesite are believed to be repositioned to coordi-nate the substrate for catalysis (19, 26). How-ever, there is still no structural evidence of theexistence of this conformational change. Thephosphodiester bond cleavage is an SN2-typetransesterification reaction, which uses a two-Mg2+-ion mechanism that is also used by thegroup I and group II introns as well as thespliceosome (27–30). Despite extensive studies,the positions of the catalytic Mg2+ ions and thecomposition of the catalytic center in RNase Pare still enigmatic because of the lack of struc-tural information of a pre-tRNA substrate–boundRNase P (24, 31–39). Consequently, the chemicalmechanism of this two-Mg2+-ion–mediated reac-tion still awaits to be revealed.Here, we present the 3.5-Å cryo-EM structures

of S. cerevisiae nuclear RNase P holoenzymealone and in complex with a pre-tRNA substrate.The structures unveil the arrangement and func-tion of all the subunits within yeast RNase P andprovide an integrated model that depicts how

RESEARCH

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 1 of 11

1Shanghai Institute of Precision Medicine, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China. 2State Key Laboratory of Molecular Biology, CASCenter for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (CAS), Shanghai 200031, China. 3University of ChineseAcademy of Sciences, CAS, Shanghai 200031, China. 4Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics,CAS, Dalian 116023, China. 5School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. 6Hefei National Laboratory for Physical Sciences at Microscale and Schoolof Life Sciences, University of Science and Technology of China, Hefei 230027, China. 7Key laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao TongUniversity School of Medicine, Shanghai 200025, China. 8National Facility for Protein Science in Shanghai, Zhangjiang Laboratory, Shanghai, 201210, China. 9Shanghai Science Research Center,CAS, Shanghai, 201204, China.*These authors contributed equally to this work.†Corresponding author. Email: leim@shsmu.edu.cn (M.L.); ghli@dicp.ac.cn (G.L.); wujian@shsmu.edu.cn (J.W.)

on Septem

ber 30, 2020

http://science.sciencemag.org/

Dow

nloaded from

the pre-tRNA substrate is recognized and howthe hydrolysis of the 5′-leader of pre-tRNA is cat-alyzed by eukaryotic RNase P.

Overall structure of yeast RNase P

We used a two-step affinity purification schemeto obtain the endogenous S. cerevisiae RNase Pcomplex. The highly purified RNase P was an-alyzed by means of SDS–polyacrylamide gelelectrophoresis followed by mass spectrometryconfirmation (fig. S1, A and B, and supplemen-tary materials, materials and methods). YeastRNase P exhibited a robust pre-tRNA cleavageactivity in the presence of magnesium ions,indicating the recovery of a fully functionalribozyme (fig. S1C). Single-particle EM analysisof yeast RNase P yielded a well-defined EMdensity map at an overall resolution of 3.5 Å(figs. S1, D to J, and S2). We combined de novomodel building and homologous modeling togenerate an atomic structure for the RNase Pcomplex (Fig. 1, A and B; fig. S3; and table S1).The final refined model of yeast RNase P con-tains all the previously identified components,one catalytic RNA—Rpr1—and nine proteins(Fig. 1B).The protein components of yeast RNase P

form an intimately interconnected hook-shapedarchitecture, with Pop1, Pop6, and Pop7 beingthe head and Pop3, Pop4, Pop5, Pop8, Rpp1,and Rpr2 being the arm (Fig. 1C). Structurally,the protein hook can be considered as the as-sembly of Pop1, Pop6-Pop7 heterodimer, Pop5-Pop8-(Rpp1)2 heterotetramer, and Pop4-Rpr2-Pop3heterotrimer (Fig. 1C). The Rpr1 RNA adopts anextended and slightly curved single-layered con-figuration, with the C and S domains packingagainst each other (Fig. 2, A and B). Three co-axially stacked helical stems, P2-P19 and P4-P1of the C domain and P8-P9 of the S domain,form the core of the RNA, which is covered byPop1 on one side and by the Pop5-Pop8-(Rpp1)2heterotetramer on the other (Fig. 1B). In thehead module of the hook, the Pop6-Pop7 het-erodimer together with Pop1 encircles the P3branch of Rpr1 (Fig. 1B). The arm of the hookpacks along one side of Rpr1 (Fig. 1B). At theend of the hook, the Pop4-Rpr2-Pop3 hetero-trimer functions as a bridge between the C andS domains of Rpr1 (Fig. 1B). Together, the pro-tein hook tightly wraps around the Rpr1 RNA,burying a total of ~14,715 Å2 surface area be-tween the RNA and the proteins (Fig. 1B).

Structure of the Rpr1 RNA

In the coaxial helical core of Rpr1, three highlyconserved regions—CR-I, CR-IV, and CR-V—foldtogether into a distinct pseudo-knot structure(Fig. 2, A and C). This pseudo-knot structurebrings the P2-P19 stem into close vicinity tostem P4-P1 so that the two long stems packparallel to each other (Fig. 2A). By contrast,stem P8-P9 in the S domain is not close to stemP4-P1 in primary sequence (Fig. 2B). Both theterminal loops of stems P8 and P9 adopt atetraloop conformation and respectively inter-act with the minor grooves of stems P4 and P1

through tetraloop-tetraloop receptor interac-tions (Fig. 2, A and D). Beside CR-I, CR-IV, andCR-V, there are two additional universally con-served regions CR-II and CR-III, correspondingto two single-stranded junctions J11/12 andJ12/11, between stems P10/11 and P12 in the Sdomain (Fig. 2B). J11/12 and J12/11 fold intotwo interleaving T-loop motifs that are stabilizedby an intricate network of non–Watson-Crickinteractions among the conserved nucleotides(Fig. 2E).The most striking feature of yeast RNA is that

six stems (P3, P7, P8′, P10/11, P12, and P15) areloosely connected to the helical core, expandingoutward from the RNA center (Fig. 2A). Thisexpanded conformation of Rpr1 yields three bigopen holes in the RNA structure (Fig. 2A). Insharp contrast, RNAs of bacterial RNase Psadopt much more compact, two-layered config-urations, with auxiliary elements that mediatelong-range interactions among different RNAregions to ensure the correct fold and the stab-ility of the RNA (fig. S5, A and B) (23, 40, 41).Another difference between bacterial and yeast

RNase P RNAs is from the P3 branch. In bacteriaRNase P RNA, the P3 branch is a continuousbase-paired stem (fig. S4, A and B). By contrast,the P3 branch of yeast Rpr1 contains a large un-paired region that separates the P3 branch intotwo helical stems (Fig. 2, A and B). Hereafter, wewill refer to the proximal and distal helical stemsas P3 and P3′, respectively, and the two unpairedsingle-stranded regions between stems P3 and

P3′ as junctions J3/3′ and J3′/3, respectively(Fig. 2, A and B).

Head module of the protein hook and itsinteraction with Rpr1

The largest protein subunit Pop1 is specific toeukaryotic RNase Ps (Fig. 1, A to C) (42). Fromthe N to C termini, Pop1 contains three motifs:an N-terminal motif (NTM), an internal motif(INM), and a large C-terminal globular domain(CTD) (Fig. 3A). A long helix a1 of Pop1NTM sticksout and mediates the interaction with Pop5(Fig. 3A). The rest of Pop1NTM folds into a smallhelical and highly basic structure, plugging intothe open junction between CR-IV and stems P4,P7, and P15 of Rpr1 (Fig. 3B). The side chains ofmultiple arginine residues in Pop1NTM make ex-tensive stacking and electrostatic interactionswith nucleotides in stems P4, P7 and P15, andCR-IV from four different directions (Fig. 3Band fig. S5A). These arginine residues are highlyconserved from yeast to humans (fig. S5B), sug-gesting that Pop1NTM plays an important role instabilizing the RNA structure in all eukaryoticRNase Ps. Consistent with this notion, mutationof conserved Arg97Arg98Arg99 in yeast Pop1 re-sulted in severe defect in pre-tRNA processing(43). The INM of Pop1 contains three helices anda long loop, fitting into the big open hole amongstems P7, P8′, P8, P9, and P11 and stabilizing theconformation of the S domain of Rpr1 (Fig. 3Cand fig. S5C). Pop1CTD is located at the concave sideof the Rpr1 RNA opposite to Pop1NTM (Fig. 3D).

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 2 of 11

Fig. 1. EM Structure of the S. cerevisiae RNase P holoenzyme. (A) The EM density map ofS. cerevisiae RNase P at an average resolution of 3.5 Å. The individual protein and RNA subunits ofRNase P are colored according to the scheme shown at the bottom right of the figure. (B) Overallstructure of the S. cerevisiae RNase P complex. (C) Two orthogonal views of the overall structure ofthe protein hook.

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

Three conserved basic patches in Pop1CTD me-diate interactions with different regions of Rpr1,covering a large surface area of the helical coreof Rpr1 and stabilizing the single-layered archi-tecture of the RNA (Fig. 3D and fig. S5D). Con-sistent with this notion, mutations of key residueson all three patches cause defects in pre-tRNAprocessing (fig. S5B) (43).The P3 branch of Rpr1 binds to the concaved

surface of the saddle-shaped Pop6-Pop7 hetero-dimer, with junctions J3/3′ and J3′/3 sitting onthe basic center of the saddle (fig. S6, A and B).In contrast to Pop6, which only interacts withthe P3 branch, Pop7 also contacts stem P15 andis sandwiched in the wedge between stems P3and P15 of Rpr1 (fig. S6C). Binding with Pop6-Pop7 induces an ~120° bend between the P3branch and the single-layered core of Rpr1, sothat Pop7 and Pop1 form an extensive interface

and the P3 branch of Rpr1 is encircled by Pop1,Pop6, and Pop7 (fig. S6D). Taken together, thehead module of the protein hook wraps aroundthe C domain and part of the S domain of Rpr1,stabilizing the helical RNA core as well as looselyconnected stems P3′ and P15 (Fig. 3E).

Arm module of the protein hook and itsinteraction with Rpr1

The arm module of the protein hook containstwo subcomplexes, the Pop5-Pop8-(Rpp1)2 hetero-tetramer and the Pop4-Rpr2-Pop3 heterotrimer(Fig. 1C). Pop5, Pop8, and two copies of Rpp1form a heterotetramer with a pseudo-twofoldsymmetry (Fig. 3F). The heterotetramer is con-nected with the head module through two dis-crete, reciprocal interactions with Pop1 involvingPop5 and one Rpp1 molecule (Fig. 1C). Hereafter,we will refer to the Rpp1 molecule that interacts

with Pop1 as Rpp1A and the other as Rpp1B (Fig. 1C).Although there is very limited sequence sim-ilarity between Pop5 and Pop8, their structureshighly resemble each other, with a root meansquare deviation of 2.5 Å (fig. S7A). The Pop5-Pop8-(Rpp1)2 heterotetramer sits on one sideof the C domain of Rpr1 opposite to Pop1CTD(Figs. 1B and 3G and fig. S7, B and C). The sur-face of one side of Pop5 is highly basic, forminga deep cleft that tightly holds the zig-zaggedCR-IV of Rpr1 (Fig. 3H). The highly conservedbasic N terminus of Pop5 embraces CR-IV, withthe side chains of Arg3 and Lys5 pointing into thenarrow space between CR-I, CR-IV, and CR-V(fig. S7D). In addition to Pop5, Rpp1B also co-ordinates electrostatic interactions with bothCR-I and CR-V (fig. S7B), so that Pop5 and Rpp1Btogether stabilize the pseudo-knot structure ofRpr1 by means of a large extended comple-mentary interface (Fig. 3G).This Pop4-Rpr2-Pop3 heterotrimer is connected

to the Pop5-Pop8-(Rpp1)2 heterotetramer throughextensive interactions between the b-barrel ofPop4 and the C-terminal long coiled coil of Rpp1B,so that the two subcomplexes together formthe rod-shaped arm module of the protein hook(Figs. 1C and 3I and fig. S8A). The b-barrel ofPop4 interacts with the minor groove of stemP1 of Rpr1 through electrostatic interactions(Fig. 3J and fig. S8B). The two ends of longhelix a3 of Pop4 respectively pack on the terminiof stems P1 and P9, stabilizing the tetraloop-tetraloop receptor interaction between stems P1and P9 (Fig. 3J and fig. S8C). Rpr2 mainly in-teracts with the S domain of Rpr1; helices a2and a3, one edge of the b-sheet, and peripheralloops of Rpr2 constitute a positively chargeddepression that holds the U-shaped T-loop ofJ12/11 of Rpr1 (Fig. 3J and fig. S8D). In contrastto Pop4 and Rpr2, Pop3 makes limited directinteractions with the RNA; only the extendedb-sheet of Pop3 contacts J11/12 of Rpr1 (Fig. 3Jand fig. S8E). Taken together, the Pop4-Rpr2-Pop3 heterotrimer functions as a bridge be-tween the C and S domains of Rpr1, stabilizingthe position of J11/12 and J12/11 relative to thehelical core of the RNA (Fig. 3K).

The protein hook stabilizes theRpr1 RNA

To corroborate our structural study, we carriedout molecular dynamics (MD) simulations toinvestigate how the protein components affectthe structural stability of the Rpr1 RNA. MDsimulation trajectories indicated that Rpr1alone exhibited a very large fluctuation in thebackbone phosphate atoms (Fig. 4A). Associa-tion of the protein hook with Rpr1 greatly sup-pressed this fluctuation except for stems P8′and P12, which make no direct contact with theproteins (Fig. 4A). Binding with Pop1 alone wasable to substantially reduce the fluctuation ofRpr1, especially the P3 branch and stem P15(Fig. 4A). Other than the structural variationof individual nucleotides, MD simulation alsoshowed that except for stems P8′ and P12, localcross-correlation in Rpr1 was suppressed upon

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 3 of 11

Fig. 2. Structure of the catalytic RNA subunit Rpr1. (A) Two orthogonal views of the overallstructure of the Rpr1 RNA. The three open holes are highlighted by the red triangle, circle, andrectangle, respectively. (B) Secondary structure diagram of the yeast Rpr1 RNA. RNA elementsare colored as in (A). The C and S domains are denoted. The canonical Watson-Crick andnoncanonical base-pairing interactions are shown as solid lines and dots, respectively. Nucleotides inCRs that are universally conserved in bacterial, archaeal, and eukaryotic RNase P RNAs arehighlighted with solid circles, whereas those that are invariant only within eukaryotic RNAs arehighlighted with open circles. (C) Structure of the universally conserved pseudo-knot formed byCR-I, R-IV, and CR-V. (D) The tetraloop-tetraloop receptor interactions between stems P4 and P8and between P1 and P9. The interacting regions are highlighted in red boxes. (E) A close-up cartoonrepresentation of the CR-II/CR-III region in the S domain.

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

protein hook association (fig. S9), suggestingthat binding with proteins reduced the relativemovements of individual structural elementswithin Rpr1 so that the RNA behaves more asa whole in the RNase P complex.To obtain an overall picture of the atomic

motions in Rpr1, we performed principle com-ponent analysis (PCA) using Cartesian coordi-nates of the phosphate atoms of Rpr1. Theprojection of trajectories onto the first twoprincipal components, PC1 and PC2, accountedfor a substantial amount of overall motion ofRpr1 in phase space. Consistent with the cross-correlation analysis, except for stems P8′ andP12, the overall motion of Rpr1 along PC1 andPC2 was greatly constrained by proteins in theRNase P complex (Fig. 4B and Movie 1). Further-more, Gibbs free-energy landscape (FEL) analysisshowed that for Rpr1 in the RNase P complex,a single global energy minimum was observed,indicating that the conformational state of Rpr1is well restricted by the protein components(Fig. 4C). In sharp contrast, many local minimum-energy basins were observed for Rpr1 alone,which is suggestive of thermodynamically lessstable conformations (Fig. 4C). Collectively, MDsimulation revealed an obvious stabilizing effecton the Rpr1 RNA by the protein hook.

pre-tRNA recognition

To gain insights into the pre-tRNA recognitionand 5′-leader processing mechanism, we deter-mined the cryo-EM structure of yeast RNase Pin complex with a yeast pre-tRNAPhe at a reso-lution of 3.5 Å (figs. S10, A to H, and S11, A toE, and table S1). In this pre-tRNA substratebound structure, the pre-tRNA substrate sits ina large open pocket on one side of the holo-enzyme (Fig. 5, A to C). The coaxially stackedacceptor and T stems of the pre-tRNA substratemake extensive intermolecular interactions withboth the Rpr1 RNA and protein components,positioning the scissile phosphate of pre-tRNAright at the active site of the ribozyme (Fig. 6Aand fig. S11, A to C). In addition, one side of theanticodon arm of pre-tRNA also mediates exten-sive contacts with Rpp1B through complementaryinterface, further stabilizing the pre-tRNA inthe binding pocket (Fig. 5, A to C).The cryo-EM structure of yeast RNase P–pre-

tRNA complex provided an atomic model ofthe “measuring device” of eukaryotic nuclearRNase Ps. The “measuring device” for pre-tRNArecognition is mainly composed of two anchorsin RNase P (Fig. 6A). At the first anchor site,two unpaired nucleotides—A204 and G245, fromthe conserved T-loops in the S domain of Rpr1—form p-p stacking interactions with two un-stacked bases, C56 from the TyC loop and G19from the D loop of the pre-tRNA, respectively(Fig. 6B and fig. S11D).At the other end of the acceptor stem, where

the scissile phosphate resides, the highly con-served N-terminal motif of Pop1 (Pop1NTM) func-tions as the second anchor to stabilize thepre-tRNA at the catalytic center in the C domainof RNase P (Fig. 6C). The N-terminal long helix

a1 in Pop1NTM that packs with Pop5 sits right onthe acceptor stem of pre-tRNA so that the loopbetween helices a1 and h1 in Pop1NTM (L1-1) packson the G1-C72 base pair of pre-tRNA, forcing the3′-tailor of pre-tRNA to adopt a sharp turn at A73and fold back to the 5′-leader (Fig. 6C). The baseof pre-tRNA A73 flips out away from the acceptorstem and is sandwiched between the base ofRpr1 G300 and the aliphatic side chain of Arg115

in Pop1NTM (Fig. 6D). The helical core of Pop1NTMalso mediates extensive hydrogen-bonding andelectrostatic interactions with the first three basepairs of the pre-tRNA (Fig. 6E).MD simulation analysis revealed a broad dis-

tribution of the distance between the two an-chor points of Rpr1, which is suggestive of avery dynamic behavior of the RNA (Fig. 6F). Inthe presence of the protein hook, this distance

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 4 of 11

Fig. 3. The protein hook and its interaction with the Rpr1 RNA. (A) Overall structure of Pop1.(B) An overall view of the interaction between Pop1NTM and the Rpr1 RNA. The RNA is shown ascartoon with semitransparent surface. CR-IV and stems P4, P7, and P15 that forms the bigopen hole are denoted. (C) An overall view of Pop1INM (colored in yellow) that fits into the big openhole among stems P7, P8′, P8, P9, and P11. (D) Interactions between Pop1CTD and Rpr1. (E) Thehead module of the protein hook wraps around the C domain and part of the S domain of Rpr1,stabilizing stems P3′ and P15. (F) Overall structure of the Pop5-Pop8-(Rpp1)2 heterotetramer.(G) The Pop5-Pop8-(Rpp1)2 heterotetramer sits on one side of the C domain of Rpr1, mediatingextensive interactions with the pseudo-knot. (H) Analysis of the electrostatic surface potential ofPop5 reveals a highly basic deep cleft that holds CR-IV tightly (red, negative; blue, positive).(I) Overall structure of the Pop4-Rpr2-Pop3 heterotrimer. (J) Overall view of the interaction betweenthe Pop4-Rpr2-Pop3 heterotrimer and the Rpr1 RNA. (K) The Pop4-Rpr2-Pop3 heterotrimerfunctions as a bridge between the C and S domains of Rpr1.

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

is tightly constrained to ~51 Å and is optimalto accommodate the coaxially stacked acceptorstem and T-stem of pre-tRNAs, which have afixed length of ~45 Å from the cleavage site tothe TyC loop (Fig. 6G). The Pop4-Rpr2-Pop3heterotrimer by itself already had a strongstabilizing effect (Fig. 6F), which is consistentwith this trimer functioning as a bridge be-tween the C and S domains, stabilizing theposition of the T-loops relative to the catalyticcenter (Fig. 5, B and C). Taken together, ourstructural and MD simulation results suggestedthat the protein hook stabilizes yeast RNase Pinto a “measuring device,” with two fixed anchorsthat recognize the L-shaped structure rather thanspecific sequences of pre-tRNA substrates (1).

5′-leader recognition

In the complex structure, at least four nucleo-tides in the 5′-leader of pre-tRNA were visiblein the cryo-EM density map (fig. S12A). Therecognition of the 5′-leader involves both theRpr1 RNA and protein subunits. Pop5 and Rpp1Bform a continuous basic surface that holds the5′-leader (Fig. 6H). The 5′ terminal nucleotideA(–4) of pre-tRNA sticks into a hydrophobicpocket between Pop5 and Rpp1B, with its basestacking with the side chain of Phe140 of Rpp1B(Fig. 6I). Nucleotides at the –1 and –2 positionsof pre-tRNA—A(–2) and A(–1), respectively—sequentially stack on G1 following the double-stranded trajectory of the acceptor stem (Fig. 6I).This continuous base-stacking is extended intonucleotide A314 in the zig-zagged turn of CR-IVin Rpr1 (Fig. 6I). Nucleotide G(–3) of the 5′-leaderdiverges from this base stack and packs on C313of Rpr1 (Fig. 6I). In addition to these stackinginteractions, G(–3) and A(–2) also make multiplehydrogen-bonding interactions with C313 andA314 of Rpr1 (fig. S12B), further strengtheningthe connection between the 5′-leader of pre-tRNA and CR-IV of Rpr1. The zig-zagged turnof CR-IV is tightly grabbed by a highly basiccleft of Pop5, emphasizing that one of the majorfunctions of Pop5 is to stabilize CR-IV of Rpr1 for5′-leader recognition. In addition to this indirectrecognition of the 5′-leader via CR-IV of Rpr1,Pop5 also directly contacts the backbone ofnucleotides A(–1) and A(–2) of pre-tRNA throughhydrogen-bonding interactions (Fig. 6I).

Active site

The location of the catalytic center is inferredfrom the scissile phosphate of the pre-tRNAsubstrate, which resides right on the P4 stem ofthe Rpr1 RNA (Fig. 7A). The distance betweenthe scissile phosphate and the closest proteinsubunit Pop5 is ~7 Å, arguing against proteincomponents playing any direct role in the catal-ysis (fig. S13A). By contrast, the scissile phos-phate of pre-tRNA is closely surrounded by apanel of Rpr1 nucleotides: A91, U92, andU93 fromCR-I and G343 and A344 from CR-V (Fig. 7A).Equivalent nucleotides of A91 and U92 in Bacillussubtilis and Escherichia coli RNase P RNAs havebeen implicated in metal ion coordination andcatalysis (24, 35, 36, 39, 44, 45).

A putative Mg2+ ion (M1) was identified inthe vicinity of U92 and U93 in the EM densitymap (fig. S13B). This site coincides with ametal site observed in the crystal structures ofT. maritima and Bacillus stearothermophilusRNase Ps (23, 41). The M1 Mg2+ ion is coordi-nated by inner-sphere contacts with the hydroxylof the conserved U93 in Rpr1 and three non-bridging phosphoryl oxygens, pro-Sp of Rpr1 A91,pro-Rp of Rpr1 U92, and pro-Rp of the scissilephosphate of pre-tRNA (Fig. 7A). Several lines ofevidence from previous nucleotide substitutionstudies of bacterial RNase P support this geom-etry of the M1 Mg2+ ion and its environment.First, phosphorothioate-rescue experiments showthat the pro-Rp but not Pro-Sp oxygen of G50 inB. subtilis RNase P RNA (equivalent to yeastRpr1 U92) binds one metal ion (36). Second,the 4-thiouridine (4SU) modification of U51 of

B. subtilis RNase P RNA (equivalent to yeastRpr1 U93) greatly decreases phosphodiester bondcleavage of pre-tRNA (24).A hallmark of the two–metal ion catalysis is

that the two metal ions maintain a distance of~4 Å and are located roughly in line with thephosphor-sugar backbone on the opposite sidesof the scissile phosphate through interactionswith the same nonbridging phosphoryl oxygen(27, 46). Although the local cryo-EM density mapdid not unambiguously reveal the position of thesecond metal ion, we could model a putativeMg2+ (M2) ion that is within the density mapand is in accordance with the aforementionedconstraints (fig. S13B). The M2 Mg2+ ion is co-ordinated by nonbridging phosphoryl oxygensof A91, G343, and A344 of Rpr1 and the O3′leaving group of A(–1) in the pre-tRNA substrate(Fig. 7A). Consistently, previous biochemicalstudies have implicated major constituents ofthe M2 site in B. subtilis RNase P RNA, pro-Spoxygen of A50, and pro-Rp of A390 (equivalentto yeast Rpr1 U92 and G344, respectively), indirect inner-sphere coordination of a Mg2+ ionimportant for RNase P activity (32, 36, 38, 39).Taken together, the structure of the catalyticcenter in yeast RNase P is in perfect agreementwith previous biochemical data of bacterial RNase P,suggesting that the configuration of the RNA-based catalytic center is universally conserved inall RNase Ps, from bacteria to eukarya.

Substrate-induced activation of RNase P

Previous kinetic studies suggested that a con-formational change may occur after the forma-tion of the initial RNase P–pre-tRNA complexand before the cleavage step to form the product(18, 19, 25, 47, 48). To examine this conformationalchange, we superimposed the aporibozyme (apo)and pre-tRNA–bound yeast RNase P structuresand found a dramatic conformational change inthe active center of Rpr1, although the overallstructure of the RNA remains largely unchanged(Fig. 7, B andC; fig. S14, A and B; andMovie 2). Inthe presence of pre-tRNA, the N-terminal tail ofPop5 rotates toward the catalytic center to formtwo hydrogen bonds with nucleotide A(–2) inpre-tRNA, helping stabilize the 5′-leader in theactive site (Fig. 7B and fig. S14, A and B). Con-sequently, the conformation of nucleotides G343and A344 in Rpr1 is adjusted to be optimal forcoordinating a catalytic Mg2+ ion (M2) (Fig. 7Band fig. S14, A and B). The most prominent con-formational change is from nucleotide U93 ofRpr1. Upon binding of pre-tRNA, U93 is forcedto rotate a large angle from a position outside ofstem P4 to point into the catalytic center andcoordinate the other catalytic Mg2+ ion (M1)(Fig. 7, B and C, and fig. S14, A and B). The po-sition of U93 in the active center is furtherstabilized by the C94-G349 base pair in stem P4of Rpr1 through hydrogen-bonding interactions(Fig. 7D). MD simulation analysis revealed thatin the apo-ribozyme, U93 bulges out of the P4stem of Rpr1 and exhibits a dynamic conforma-tion (fig. S15). In sharp contrast, binding of pre-tRNA tightly constrains U93 in a fix orientation

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 5 of 11

Movie 1. The first two essential modes ofRpr1 obtained from PCA using MDsimulation trajectories. (Left) Absent of theprotein components. (Right) Present of theprotein components.

Movie 2. Local conformational change in theactive center of RNase P upon pre-tRNAbinding.The morphed trajectory was generatedthrough linearly interpolating the conformationsof the Rpr1 RNA between the apo and pre-tRNA–bound states of yeast RNase P based onthe cryo-EM structures. Although the inter-mediates are unphysical, the movie clearlydemonstrates the major conformational differ-ences of Rpr1 upon pre-tRNA binding.

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

that precisely matches the conformation ob-served in the complex structure (fig. S15). Takentogether, comparative analysis of the apo andpre-tRNA–bound structures of yeast RNase Prevealed aMg2+ ion–dependent substrate inducedconformational change in the catalytic center,

which transforms the ribozyme froman inactiveinto an active state.

Catalytic mechanism

On the basis of the structure of the active site,we proposed a catalytic mechanism of pre-tRNA

cleavage by RNase P. In this model, the M1 Mg2+

ion coordinates the pro-Sp oxygen of the scissilephosphate and an attacking nucleophilic watermolecule (Fig. 8A). The catalytic role for the M1Mg2+ ion is to lower the pKa value of the nu-cleophilic water (where Ka is the acid dissociation

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 6 of 11

Fig. 5. Overall structure ofthe S. cerevisiae RNase Pholoenzyme complexed withpre-tRNAPhe. (A) Overallview of the EM density map ofS. cerevisiae RNase Pcomplexed with pre-tRNAPhe.The individual protein and RNAsubunits of RNase P and thepre-tRNA substrate are coloredaccording to the schemeshown at the right of the figure.A close-up view of the activecenter bound with the pre-tRNA substrate is shown onthe right. (B to C) Overallstructure of the S. cerevisiaeRNase P–pre-tRNA complex.The protein components are incartoon (B) or surface (C)representation. The RNAcomponent and pre-tRNAare shown in cartoon.

Fig. 4. The protein hookstabilizes the Rpr1 RNA.(A) The root mean squarefluctuation (RMSF) ofbackbone phosphorousatoms of the Rpr1 RNA alone(black), in the presence ofall protein components (red),or only Pop1 (cyan), thePop6-Pop7 heterodimer(magenta), the Pop5-Pop8-(Rpp1)2 heterotetramer(blue), or the Pop4-Rpr2-Pop3 heterotrimer (green).(B) Porcupine plots of thefirst two eigenvectors gener-ated by means of PCA. Thevectors represented as redarrows illustrate the ten-dency of the movement ofthe Rpr1 RNA in the absence(left) or in the presence(right) of the protein compo-nents. (C) Free-energy land-scape calculated byprojecting the conforma-tional space onto the twoprincipal components(PC1 and PC2) in theabsence (top) or inthe presence (bottom) ofthe protein components.

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

constant and pKa = –log10Ka), thus facilitatingthe deprotonation of the water molecule togenerate a hydroxide ion that performs an SN2in-line attack on the phosphodiester bond witha trigonal bipyramidal transition state (Fig. 8A).The M2 Mg2+ ion coordinates the same pro-Spoxygen of the scissile phosphate as well as theupstream ribose O3′ oxygen atom, playing astabilizing role in maintaining the geometry ofthe transition state and the leaving 5′-leader(Fig. 8A).We used multiscale quantum mechanical/

molecular mechanical (QM/MM) free-energysimulations to unveil the underlying catalyticmechanism of yeast RNase P. First, we performed250-ns classical MD simulations to evaluate thestructural arrangement of the catalytic center,where a putative nucleophilic water molecule isproposed to coordinate the M1 Mg2+ ion (fig. S16).The key distance profiles (d1 to d7) of the activesite obtained from classical MD simulations are

quantitatively consistent with the structural ar-chitecture of the catalytic site observed in thecryo-EM structure (fig. S16).Next, we carried out QM/MM simulations,

combining umbrella samplings and path collec-tive variables (CVs), to obtain the free-energyprofile of the phosphodiester cleavage reactionof pre-tRNA. A path connecting the reactantand product in a seven-CV space (distance d1to d7) was used to explicitly define the progressof the reaction. The free-energy profile alongthe converged minimum free-energy path (MFEP)revealed a metastable intermediate (INT) regionseparated by two transition states (TS1 and TS2)(Fig. 8B). After crossing the TS1 by overcominga free-energy barrier of ~18.56 kcal/mol, thefree-energy profile exhibits an intermediateregion, corresponding to the inversion of stereoconfiguration of the pentacovalent phosphoraneintermediate (Fig. 8B). Then, the reaction pro-ceeds to cross the rate-limiting step TS2 with a

free-energy barrier of ~18.75 kcal/mol (Fig. 8B).The calculated free-energy barrier is in excellentagreement with the experimentally measuredrate constants of 0.35 ± 0.03 s−1, correspond-ing to a Gibbs free energy change (DG‡) value of~18.19 kcal/mol (25).On the basis of the simulations, we depicted

the details of the catalytic mechanism. (i) Theearly stage of the MFEP starts with a decreaseof the distance between the scissile phosphateand the nucleophile water (d2), indicating thebeginning of the nucleophilic water to attackthe scissile phosphate (Fig. 8, B and C). The dis-tance between the scissile phosphate and theleaving O3′ atom (d1) exhibits a perceptible in-crease after d2 is less than 2.7 Å (path node = 3)(Fig. 8B). The distance between the M2 Mg2+

and the leaving O3′ group (d7) also decreases sub-stantially when the nucleophilic water approachesthe scissile phosphate before the TS is reached,suggesting stabilization of the leaving O3′ by the

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 7 of 11

Fig. 6. The pre-tRNAPhe substrate recogni-tion. (A) Overview of the interactions betweenthe yeast RNase P holoenzyme and the pre-tRNA substrate. Interactions at the two anchorsites are highlighted in red and blue boxes,respectively. (B) A close-up view of the stackinginteractions between the TyC and D-loops ofpre-tRNA and the T-loops in CR-II and CR-III ofthe Rpr1 RNA. (C) Two related close-up viewsof the interactions between Pop1NTM andpre-tRNA. The pre-tRNA, Pop1NTM, and thepseudoknot of Rpr1 were colored in cyan,green, and orange, respectively. (D) A close-upview of the interaction between the 3′-tailorof pre-tRNA and Pop1NTM. (E) A close-up view ofthe interactions between Pop1NTM and the firstthree base pairs in the acceptor stem of pre-tRNA. (F) Distribution of the distance betweenthe backbone phosphorus atom of A344 and theC1′ atom of G245 of Rpr1, sampled from a totalof ~1.8 ms integrated accelerated moleculardynamics simulation trajectories of eachsystem. (G) Overall view of the distancebetween the conserved A344 and G245 locatedin C and S domains of Rpr1, respectively.A pre-tRNA in ribbon representation is alsoshown. The distance between the cleavage siteand the TYC-loop is denoted. (H) Recognition ofthe 5′-leader of pre-tRNA by yeast RNase P.The protein subunits Pop5 and Rpp1B areshown in surface (left) or electrostatic surface(right) representation, respectively. Fournucleotides in the 5′-leader were colored indark pink and shown in cartoon. The rest of pre-tRNA and the conserved pseudoknot ofRpr1 were shown in cartoon and colored in cyanand orange, respectively. (I) A close-up viewof the interactions between the 5′-leaderand RNase P. The individual components ofRNase P are colored as in (H).

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

M2 Mg2+ ion. (ii) At the TS1 point, where d1equals d2, the free-energy profile reaches thefirst maximum value, and the scissile phosphateevolves into a pentavalent phosphorane-likegeometry (Fig. 8, B and C). Simultaneously, thenucleophilic water’s proton is released, and ahydroxyl group is formed after the TS1 (Fig. 8,B and C). We found that a bulk water molecule,stabilized by the nonbridging oxygen atoms ofG1 in pre-tRNA and U93 in Rpr1, serves as ageneral base to accept the nucleophile’s proton(Fig. 8, A to C). A transient hydronium ion (H3O

+)is identified to facilitate the proton transfer tothe pro-Sp oxygen of the scissile phosphate,forming a mono-anionic phosphorane interme-diate (Fig. 8, A to C). (iii) The product state isaccompanied by the formation of the new phos-phoryl bond between the nucleophile and thescissile phosphate (d2 ≈ 1.64 Å) and the depar-ture of the leaving O3′ group (d1 ≈ 3.00 Å).(iv) During the late stage of the MFEP, the free-energy profile exhibits a downhill process, andthe stereo configuration at the phosphorus isinverted, indicating a typical in-line SN2 nucle-ophilic substitution reaction mechanism (Fig. 8,B and C). We observed that the proton that istransferred onto the pro-Sp oxygen of the scissilephosphate at TS2 is spontaneously shuttledtoward the leaving O3′ group after the inver-sion of the configuration (Fig. 8, B and C).

Discussion

The catalytic mechanism of how RNase P facil-itates the cleavage of pre-tRNA is a central ques-tion in the RNase P field. Our structural andsimulation data reported here provide an in-tegrated mechanistic insight into the RNase P–catalyzed pre-tRNA processing (Fig. 8D). Ourdata reveal that the RNase P holoenzyme islargely preassembled to engage the pre-tRNAsubstrate through a two-anchor mechanism(Fig. 6A). One of the anchors, the T-loops inthe S domain of Rpr1, that recognizes the TyCand D loops of pre-tRNA is universally conserved,whereas the other that recognizes the pre-tRNAcleavage site has evolved from an RNA-basedapparatus in bacteria to a protein-based one ineukaryotic RNase Ps (Fig. 6C) (23). The distancebetween the two anchors is optimal for accom-modating the coaxially stacked acceptor andT stems of pre-tRNA substrates (Fig. 6F), sug-gesting that these anchor sites function as the“measuring device” to recognize the pre-tRNAsubstrate. We propose that it is this “measuringdevice” of all RNase P complexes that mediatesthe initial engagement with pre-tRNA to formthe low-affinity ES complex (Fig. 8D).The pre-tRNA–bound RNase P structure re-

veals that the recognition of the 5′-leader ofpre-tRNA involves both Rpr1 and the proteinsubunit Pop5. A highly basic cleft on the sur-face of Pop5 tightly holds a zig-zagged turn inCR-IV of Rpr1 that mediates both stacking andhydrogen-bonding interactions with the 5′-leader(Fig. 6, H and I, and fig. S12B). Bacterial soleprotein subunit Rpp also contains a similar highlybasic cleft that holds CR-IV of the bacterial RNA

in the same fashion (fig. S17) (23). Therefore, it islikely that the 5′-leader of pre-tRNA is recognizedby bacterial RNase P through the same mecha-nism as in yeast RNase Ps. Unlike the 5′-leader,the 3′-tailor of pre-tRNA only makes a limitedcontribution to the recognition by yeast RNase P(Fig. 6D), which is consistent with the fact thatthe 3′-CCA sequence in eukaryotic tRNA isadded posttranscriptionally (49). This is in sharpcontrast to bacterial tRNA, in which the 3′-CCAis transcriptionally encoded and serves as a keyelement recognized by bacterial RNase P throughbase-pairing with nucleotides in the L15 loop ofRNase P RNA (23).A comparison of the apo and pre-tRNA–bound

structures of yeast RNase P indicates that afterthe initial recognition of pre-tRNA by the “mea-suring device” of RNase P, the pre-tRNA sub-strate induces a local conformation change in thecatalytic center, so that the phosphate oxygen–rich cleft near the P4 stem together with thescissile phosphate and the O3′ leaving groupof pre-tRNA coordinate two catalytic Mg2+ ionsto form the active state that is primed for ca-talysis (Fig. 7, A and B). In particular, universallyconserved U93 undergoes a dramatic conforma-tional change to mediate an inner-sphere coor-dination with the catalytically important M1 Mg2+

ion, helping to properly position the local pre-tRNA structure at the active site (Fig. 7B). Con-sistent with our data, previous studies suggestedthat nucleotide U51 in B. subtilis RNase P (equiv-

alent to U93 in yeast Rpr1) contributes to a di-valent metal ion–dependent conformational changeduring the RNase P–catalyzed reaction (18, 24).We proposed that this pre-tRNA–induced Mg2+

ion–dependent conformational change in thecatalytic center of RNase P corresponds to theconformational change between the initial EScomplex and the cleavage step. Consequently, theactive site observed in the pre-tRNA–bound com-plex structure could represent the active ES*state or a transition configuration between ESand ES* states when the M2 ion is still notfully stabilized in the catalytic center (Fig. 8D)(18–20). We further speculated that the con-formation of the initial ES complex should belargely identical to that of the ES complexexcept for the catalytic center, which insteadadopts the conformation of the active site inthe apo structure of RNase P (Fig. 8D).Our EM structure of the pre-tRNA–bound

RNase P complex revealed the configuration ofthe catalytic center of RNase P in the presenceof a pre-tRNA substrate (Fig. 7A). On the basisof this structural information, our QM/MM sim-ulations visualized the mechanistic details ofphosphodiester bond hydrolysis of pre-tRNA,which is a two-Mg2+-ion–mediated SN2 reaction(Fig. 8C). Our simulations also reveal a two-stepproton transfer pathway from the nucleophilicwater to the leaving O3′ group that involves aneighboring bulk water molecule during thecleavage process (Fig. 8, B and C). A similar

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 8 of 11

Fig. 7. Configuration of the active site and the conformational change of the catalytic centerinduced through pre-tRNA binding. (A) (Left) An overall illustration of the catalytic center of theyeast RNase P. The Rpr1 RNA and pre-tRNA are colored in orange and cyan, respectively. Mg2+ ions(M1 and M2) are shown as green spheres. (Right) A close-up view of the catalytic center. TheRpr1 RNA and pre-tRNA are colored in white and cyan, respectively.The coordination of the two Mg2+

ions are highlighted in magenta dashed lines. (B) Comparison of the active center between theapo and pre-tRNA–bounded states of yeast RNase P. Pop5, pre-tRNA, and Rpr1 in the pre-tRNA–bound state are colored in slate, cyan, and silver, respectively, and Pop5 and Rpr1 in apo RNase Pare colored in green and yellow, respectively. Conformational changes of Rpr1 U93 and the Nterminus of Pop5 are denoted. (C) The RMSF of Rpr1 nucleotides along the morphed trajectory,which is generated through linearly interpolating the conformations between the apo and pre-tRNA–bound states of Rpr1 based on the cryo-EM structures. (D) A close-up view of the detailedinteractions among the M1 Mg2+ ion, Arg99 of Pop1NTM, and the C94-G349 base-pair of Rpr1.

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

bulk water that facilitates the proton transferprocess has also been suggested in the cleavagereaction catalyzed by group II introns (50).The M2 site in our structure is different from

the second Mg2+ (TmM2) site identified in the

crystal structure of T. maritima RNase P incomplex with a mature tRNA (fig. S18) (23). TheTmM2 site was located in experiments in whichthe 5′-leader was soaked into the crystals in thepresence of Sm3 (23). In contrast to the M1 and

M2 sites in our structure, the geometry of thetwoMg2+ ions in the T.maritimaRNase P–tRNAcomplex is not consistent with the octahedralMg2+ coordination geometry (27, 51). In addition,soaking a 5′-leader in a mature tRNA-bound

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 9 of 11

Fig. 8. Catalytic mechanism of pre-tRNA processingcatalyzed by yeast RNase P. (A) Snapshot of the reactantstate showing eight distances d1 to d8 that are used to describethe progress of the reaction. The Rpr1 RNA and pre-tRNAare colored in white and cyan, respectively. Mg2+ ions (M1 andM2) are shown as green spheres. The nucleophile water (W1)and the bulk water (W2) that serves as a general base to acceptthe nucleophile’s proton are shown in stick model and sphere,respectively. W1 was modeled in the catalytic center based onthe two-metal ion mechanism, and W2 was the result of theQM/MM simulation. The rest of the catalytic center—includingMg2+ ions, Rpr1, and pre-tRNA—was based on the EM densitymap. (B) (Left) Bond distance changes monitored along theMFEP during the QM/MM free-energy simulations. Distances d1to d8 are defined as in (A). d8 is the distance between theleaving proton and the nucleophilic water oxygen. (Right) Free-energy profile along the MFEP. The reactant state (R), transitionstate 1 (TS1), transition state 2 (TS2), and product formationstate (P) are highlighted with colored areas. (C) Proposedproton transfer pathway and two-metal-ion mechanism of thephosphodiester bond cleavage reaction of pre-tRNA catalyzedby yeast RNase P. (D) Kinetic model of RNase P–mediated pre-tRNAprocessing. RNase P first recognizes the pre-tRNA substrate through adouble-anchor mechanism to form the initial low-affinity ES complex.Binding of pre-tRNA then induces a local conformational change in thecatalytic center of RNase P. In particular, nucleotide U93 within stem P4 of theRpr1 RNA undergoes a conformational change to mediate an inner-sphere

coordination of the catalytically important Mg2+ ion, so that the EScomplex is isomerized to the active ES* state. The activated ES* complexthen catalyzes the phosphodiester bond cleavage of pre-tRNA througha 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.

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

complex does not represent a pre-tRNA sub-strate–bound state. Superposition analysis showedthat the soaked 5′-leader did not occupy the sameposition as the unprocessed 5′-leader in the yeastRNase P–pre-tRNA complex (fig. S17), arguing thatthe TmM2 site in the T. maritima RNase P–tRNAcomplex is not a site for a catalytic metal ion.The cryo-EM structures reported here rep-

resent a major step forward for mechanisticunderstanding of the RNase P function. Ourdata support that all RNase P ribozymes sharean RNA-based, substrate-induced catalytic mech-anism of pre-RNA processing. Whereas bacterialRNase P RNA is catalytically active by itself, eu-karyotic RNase P is a protein-controlled ribozyme;its protein components not only directly partici-pate in substrate recognition but also stabilize thecatalytic RNA in a conformation optimal for pre-tRNA binding and cleavage reaction.

Methods and materials summary

Yeast strain constructions and affinity purifi-cation of the RNase P holoenzyme were performedas described previously, with minor modifica-tions (16). Briefly, protein subunits Pop4 andRpr2 were tagged with protein A and 3×Flag,respectively. A two-step affinity purification schemewas used to purify yeast RNase P. For substrate-bound structural studies, the in vitro transcribedyeast pre-tRNAPhe was mixed with the highlypurified yeast RNase P at 4°C and immediatelysubjected to cryo-EM grid preparation. For cryo-EM data collection and processing, the specimenswere first prepared by using a lacey carbon filmwith a continuous ultrathin carbon film. Im-ages were taken on a Gatan K2 summit cameramounted on an FEI Titan Krios electron mi-croscope operated at 300 kV. Image processingincludes motion correction, CTF estimation, par-ticles classification, and refinement. De novoatomic model building, rigid docking of knownstructures, and homologous structure modelingwere combined to generate the atomic modelfor the entire S. cerevisiae RNase P holoenzyme.QM/MM free-energy simulation was used to in-vestigate the catalytic reaction of yeast RNase P.Detailed descriptions for all the materials andmethods are provided in the supplementarymaterials.

REFERENCES AND NOTES

1. S. Altman, L. A. Kirsebom, in Ribonuclease P: In The RNA WorldR. Gesteland, T. Cech, J. Atkins, Eds. (Cold Spring HarborLaboratory Press, ed. 2, 1999), chap. 14, pp. 351–378.

2. O. Esakova, A. S. Krasilnikov, Of proteins and RNA: The RNaseP/MRP family. RNA 16, 1725–1747 (2010). doi: 10.1261/rna.2214510; pmid: 20627997

3. D. Evans, S. M. Marquez, N. R. Pace, RNase P: Interface of theRNA and protein worlds. Trends Biochem. Sci. 31, 333–341(2006). doi: 10.1016/j.tibs.2006.04.007; pmid: 16679018

4. S. C. Walker, D. R. Engelke, Ribonuclease P: The evolution of anancient RNA enzyme. Crit. Rev. Biochem. Mol. Biol. 41, 77–102(2006). doi: 10.1080/10409230600602634; pmid: 16595295

5. T. Pan, Higher order folding and domain analysis of theribozyme from Bacillus subtilis ribonuclease P. Biochemistry34, 902–909 (1995). doi: 10.1021/bi00003a024;pmid: 7827048

6. A. Loria, T. Pan, Domain structure of the ribozyme fromeubacterial ribonuclease P. RNA 2, 551–563 (1996).pmid: 8718684

7. L. Sun, F. E. Campbell, N. H. Zahler, M. E. Harris, Evidencethat substrate-specific effects of C5 protein lead touniformity in binding and catalysis by RNase P. EMBO J. 25,3998–4007 (2006). doi: 10.1038/sj.emboj.7601290;pmid: 16932744

8. S. Niranjanakumari, T. Stams, S. M. Crary, D. W. Christianson,C. A. Fierke, Protein component of the ribozymeribonuclease P alters substrate recognition by directlycontacting precursor tRNA. Proc. Natl. Acad. Sci. U.S.A. 95,15212–15217 (1998). doi: 10.1073/pnas.95.26.15212;pmid: 9860948

9. L. Sun, M. E. Harris, Evidence that binding of C5 protein toP RNA enhances ribozyme catalysis by influencing active sitemetal ion affinity. RNA 13, 1505–1515 (2007). doi: 10.1261/rna.571007; pmid: 17652407

10. Y. Kouzuma et al., Reconstitution of archaeal ribonucleaseP from RNA and four protein components. Biochem. Biophys.Res. Commun. 306, 666–673 (2003). doi: 10.1016/S0006-291X(03)01034-9; pmid: 12810070

11. H. Y. Tsai, D. K. Pulukkunat, W. K. Woznick, V. Gopalan,Functional reconstitution and characterization of Pyrococcusfuriosus RNase P. Proc. Natl. Acad. Sci. U.S.A. 103,16147–16152 (2006). doi: 10.1073/pnas.0608000103;pmid: 17053064

12. A. Terada, T. Honda, H. Fukuhara, K. Hada, M. Kimura,Characterization of the archaeal ribonuclease P proteins fromPyrococcus horikoshii OT3. J. Biochem. 140, 293–298 (2006).doi: 10.1093/jb/mvj144; pmid: 16829535

13. W.-Y. Chen, D. K. Pulukkunat, I. M. Cho, H.-Y. Tsai, V. Gopalan,Dissecting functional cooperation among protein subunits inarchaeal RNase P, a catalytic ribonucleoprotein complex.Nucleic Acids Res. 38, 8316–8327 (2010). doi: 10.1093/nar/gkq668; pmid: 20705647

14. A. Perederina, O. Esakova, C. Quan, E. Khanova,A. S. Krasilnikov, Eukaryotic ribonucleases P/MRP: The crystalstructure of the P3 domain. EMBO J. 29, 761–769 (2010).doi: 10.1038/emboj.2009.396; pmid: 20075859

15. C. W. Chan, B. R. Kiesel, A. Mondragón, Crystal structure ofhuman Rpp20/Rpp25 reveals quaternary level adaptationof the alba scaffold as structural basis for single-stranded RNAbinding. J. Mol. Biol. 430, 1403–1416 (2018). doi: 10.1016/j.jmb.2018.03.029; pmid: 29625199

16. K. Hipp, K. Galani, C. Batisse, S. Prinz, B. Böttcher, Modulararchitecture of eukaryotic RNase P and RNase MRP revealedby electron microscopy. Nucleic Acids Res. 40, 3275–3288(2012). doi: 10.1093/nar/gkr1217; pmid: 22167472

17. E. Kikovska, N.-E. Mikkelsen, L. A. Kirsebom, The naturallytrans-acting ribozyme RNase P RNA has leadzyme properties.Nucleic Acids Res. 33, 6920–6930 (2005). doi: 10.1093/nar/gki993; pmid: 16332695

18. J. Hsieh, C. A. Fierke, Conformational change in the Bacillussubtilis RNase P holoenzyme—pre-tRNA complex enhancessubstrate affinity and limits cleavage rate. RNA 15, 1565–1577(2009). doi: 10.1261/rna.1639409; pmid: 19549719

19. J. Hsieh et al., A divalent cation stabilizes the activeconformation of the B. subtilis RNase P x pre-tRNA complex:A role for an inner-sphere metal ion in RNase P. J. Mol. Biol.400, 38–51 (2010). doi: 10.1016/j.jmb.2010.04.050;pmid: 20434461

20. B. P. Klemm et al., The diversity of ribonuclease P: Protein andRNA catalysts with analogous biological functions.Biomolecules 6, 27 (2016). doi: 10.3390/biom6020027;pmid: 27187488

21. J. A. Beebe, C. A. Fierke, A kinetic mechanism for cleavage ofprecursor tRNA(Asp) catalyzed by the RNA component ofBacillus subtilis ribonuclease P. Biochemistry 33, 10294–10304(1994). doi: 10.1021/bi00200a009; pmid: 7520753

22. A. Mondragón, Structural studies of RNase P. Annu. Rev.Biophys. 42, 537–557 (2013). doi: 10.1146/annurev-biophys-083012-130406; pmid: 23654306

23. N. J. Reiter et al., Structure of a bacterial ribonuclease Pholoenzyme in complex with tRNA. Nature 468, 784–789(2010). doi: 10.1038/nature09516; pmid: 21076397

24. X. Liu, Y. Chen, C. A. Fierke, Inner-sphere coordination ofdivalent metal ion with nucleobase in catalytic RNA.J. Am. Chem. Soc. 139, 17457–17463 (2017). doi: 10.1021/jacs.7b08755; pmid: 29116782

25. J. Hsieh, S. C. Walker, C. A. Fierke, D. R. Engelke, Pre-tRNAturnover catalyzed by the yeast nuclear RNase P holoenzymeis limited by product release. RNA 15, 224–234 (2009).doi: 10.1261/rna.1309409; pmid: 19095620

26. J. C. Kurz, C. A. Fierke, The affinity of magnesium binding sitesin the Bacillus subtilis RNase P x pre-tRNA complex is

enhanced by the protein subunit. Biochemistry 41, 9545–9558(2002). doi: 10.1021/bi025553w; pmid: 12135377

27. T. A. Steitz, J. A. Steitz, A general two-metal-ion mechanismfor catalytic RNA. Proc. Natl. Acad. Sci. U.S.A. 90, 6498–6502(1993). doi: 10.1073/pnas.90.14.6498; pmid: 8341661

28. M. R. Stahley, S. A. Strobel, Structural evidence for a two-metal-ion mechanism of group I intron splicing. Science 309,1587–1590 (2005). doi: 10.1126/science.1114994;pmid: 16141079

29. N. Toor, K. S. Keating, S. D. Taylor, A. M. Pyle, Crystal structureof a self-spliced group II intron. Science 320, 77–82 (2008).doi: 10.1126/science.1153803; pmid: 18388288

30. M. R. Stahley, S. A. Strobel, RNA splicing: Group I intron crystalstructures reveal the basis of splice site selection and metalion catalysis. Curr. Opin. Struct. Biol. 16, 319–326 (2006).doi: 10.1016/j.sbi.2006.04.005; pmid: 16697179

31. W. D. Hardt, J. M. Warnecke, V. A. Erdmann, R. K. Hartmann,Rp-phosphorothioate modifications in RNase P RNA thatinterfere with tRNA binding. EMBO J. 14, 2935–2944 (1995).doi: 10.1002/j.1460-2075.1995.tb07293.x; pmid: 7540978

32. M. E. Harris, N. R. Pace, Identification of phosphates involvedin catalysis by the ribozyme RNase P RNA. RNA 1, 210–218(1995). pmid: 7585250

33. J. M. Warnecke, J. P. Fürste, W. D. Hardt, V. A. Erdmann,R. K. Hartmann, Ribonuclease P (RNase P) RNA is convertedto a Cd2+-ribozyme by a single Rp-phosphorothioatemodification in the precursor tRNA at the RNase P cleavagesite. Proc. Natl. Acad. Sci. U.S.A. 93, 8924–8928 (1996).doi: 10.1073/pnas.93.17.8924; pmid: 8799129

34. Y. Chen, X. Li, P. Gegenheimer, Ribonuclease P catalysisrequires Mg2+ coordinated to the pro-RP oxygen of the scissilebond. Biochemistry 36, 2425–2438 (1997). doi: 10.1021/bi9620464; pmid: 9054547

35. E. L. Christian, N. M. Kaye, M. E. Harris, Helix P4 is a divalentmetal ion binding site in the conserved core of theribonuclease P ribozyme. RNA 6, 511–519 (2000).doi: 10.1017/S1355838200000042; pmid: 10786842

36. S. M. Crary, J. C. Kurz, C. A. Fierke, Specific phosphorothioatesubstitutions probe the active site of Bacillus subtilisribonuclease P. RNA 8, 933–947 (2002). doi: 10.1017/S1355838202025025; pmid: 12166648

37. E. L. Christian, K. M. J. Smith, N. Perera, M. E. Harris, The P4metal binding site in RNase P RNA affects active site metalaffinity through substrate positioning. RNA 12, 1463–1467(2006). doi: 10.1261/rna.158606; pmid: 16822954

38. A. V. Kazantsev, A. A. Krivenko, N. R. Pace, Mapping metal-binding sites in the catalytic domain of bacterial RNase P RNA.RNA 15, 266–276 (2009). doi: 10.1261/rna.1331809;pmid: 19095619

39. E. L. Christian, N. M. Kaye, M. E. Harris, Evidence for apolynuclear metal ion binding site in the catalytic domain ofribonuclease P RNA. EMBO J. 21, 2253–2262 (2002).doi: 10.1093/emboj/21.9.2253; pmid: 11980722

40. A. Torres-Larios, K. K. Swinger, T. Pan, A. Mondragón,Structure of ribonuclease P—a universal ribozyme. Curr. Opin.Struct. Biol. 16, 327–335 (2006). doi: 10.1016/j.sbi.2006.04.002; pmid: 16650980

41. A. V. Kazantsev et al., Crystal structure of a bacterialribonuclease P RNA. Proc. Natl. Acad. Sci. U.S.A. 102,13392–13397 (2005). doi: 10.1073/pnas.0506662102;pmid: 16157868

42. E. Hartmann, R. K. Hartmann, The enigma of ribonuclease Pevolution. Trends Genet. 19, 561–569 (2003). doi: 10.1016/j.tig.2003.08.007; pmid: 14550630

43. S. Xiao et al., Functional characterization of the conservedamino acids in Pop1p, the largest common protein subunit ofyeast RNases P and MRP. RNA 12, 1023–1037 (2006).doi: 10.1261/rna.23206; pmid: 16618965

44. M. M. Getz, A. J. Andrews, C. A. Fierke, H. M. Al-Hashimi,Structural plasticity and Mg2+ binding properties of RNase PP4 from combined analysis of NMR residual dipolar couplingsand motionally decoupled spin relaxation. RNA 13, 251–266(2007). doi: 10.1261/rna.264207; pmid: 17194721

45. K. S. Koutmou et al., NMR and XAS reveal an inner-spheremetal binding site in the P4 helix of the metallo-ribozymeribonuclease P. Proc. Natl. Acad. Sci. U.S.A. 107, 2479–2484(2010). doi: 10.1073/pnas.0906319107; pmid: 20133747

46. S. Xiao, F. Scott, C. A. Fierke, D. R. Engelke, Eukaryoticribonuclease P: A plurality of ribonucleoprotein enzymes.Annu. Rev. Biochem. 71, 165–189 (2002). doi: 10.1146/annurev.biochem.71.110601.135352; pmid: 12045094

47. S. Wu et al., Transition-state stabilization in Escherichia coliribonuclease P RNA-mediated cleavage of model substrates.

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 10 of 11

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

Nucleic Acids Res. 42, 631–642 (2014). doi: 10.1093/nar/gkt853; pmid: 24097434

48. K. S. Koutmou, J. J. Day-Storms, C. A. Fierke, The RNR motif ofB. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer. RNA 17, 1225–1235(2011). doi: 10.1261/rna.2742511; pmid: 21622899

49. R. J. Maraia, T. N. Lamichhane, 3′ processing of eukaryoticprecursor tRNAs. Wiley Interdiscip. Rev. RNA 2, 362–375(2011). doi: 10.1002/wrna.64; pmid: 21572561

50. L. Casalino, G. Palermo, U. Rothlisberger, A. Magistrato, whoactivates the nucleophile in ribozyme catalysis? An answerfrom the splicing mechanism of group II introns. J. Am. Chem.Soc. 138, 10374–10377 (2016). doi: 10.1021/jacs.6b01363;pmid: 27309711

51. W. Yang, J. Y. Lee, M. Nowotny, Making and breaking nucleicacids: two-Mg2+-ion catalysis and substrate specificity.Mol. Cell 22, 5–13 (2006). doi: 10.1016/j.molcel.2006.03.013;pmid: 16600865

ACKNOWLEDGMENTS

We thank the staff members of the Electron MicroscopySystem at the National Facility for Protein Science in Shanghai(NFPS), Zhangjiang Laboratory, China, and the Core Facilitiesfor Protein Science at the Institute of Biophysics (IBP), ChineseAcademy of Sciences, for providing technical support andassistance in data collection. We thank M. Cao for his help ondata collection and analysis. Mass spectrometry experimentswere performed at NFPS. Funding: This work was supported bygrants from the National Natural Science Foundation of China(31525007 to M.L. and 21625302 to G.L.) and the StrategicPriority Research Program of the Chinese Academy of Sciences(XDB08010201 to M.L.). Author contributions: M.L., P.L., G.L.,J.W., and H.C. conceived the study. M.L. supervised the wholeproject. P.L., S.N., and J.C. purified the yeast RNase P complex.P.L., M.T., X.W., and S.S. prepared the cryo-EM sample. M.T.,P.L., X.W., S.Q., and G.C. collected the EM micrographs and

processed the data. J.W. and M.L. built the atomic model. Y.Z.,G.L., and X.P. performed the MD and QM/MM simulationanalyses. M.L. and P.L. wrote the manuscript. Competinginterests: The authors declare no competing interests. Dataand materials availability: The accession numbers for thestructure reported in this paper are PDB: 6AGB and 6AH3 andEMDB: EMD-9616 and EMB-9622.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/362/6415/eaat6678/suppl/DC1Materials and MethodsFigs. S1 to S18Table S1References (52–101)

22 March 2018; accepted 18 September 2018Published online 27 September 201810.1126/science.aat6678

Lan et al., Science 362, eaat6678 (2018) 9 November 2018 11 of 11

RESEARCH | RESEARCH ARTICLEon S

eptember 30, 2020

http://science.sciencem

ag.org/D

ownloaded from

Structural insight into precursor tRNA processing by yeast ribonuclease P

Peng, Gang Cai, Hong Cheng, Jian Wu, Guohui Li and Ming LeiPengfei Lan, Ming Tan, Yuebin Zhang, Shuangshuang Niu, Juan Chen, Shaohua Shi, Shuwan Qiu, Xuejuan Wang, Xiangda

originally published online September 27, 2018DOI: 10.1126/science.aat6678 (6415), eaat6678.362Science 

, this issue p. eaat6678; see also p. 644Sciencestructural basis for substrate recognition and provides insights into its catalytic mechanism.evolution. The structure of yeast RNase P in complex with its natural substrate, a tRNA precursor, demonstrates the bacterial RNA elements have been delegated to the protein components in RNase P of higher organisms duringaporibozyme structure reveals how the protein components stabilize the RNA and explains how the structural roles of

report the structures of yeast RNase P (see the Perspective by Scott and Nagai). Theet al.kingdoms of life. Now, Lan Ribonuclease P (RNase P) is a ribozyme that processes transfer RNA (tRNA) precursors and is found in all three

Structures of eukaryotic ribonuclease P

ARTICLE TOOLS http://science.sciencemag.org/content/362/6415/eaat6678

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/09/26/science.aat6678.DC1

CONTENTRELATED http://science.sciencemag.org/content/sci/362/6415/644.full

REFERENCES

http://science.sciencemag.org/content/362/6415/eaat6678#BIBLThis article cites 99 articles, 24 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.ScienceScience, 1200 New York Avenue NW, Washington, DC 20005. The title (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement ofScience

Science. No claim to original U.S. Government WorksCopyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of

on Septem

ber 30, 2020

http://science.sciencemag.org/

Dow

nloaded from