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In vitro reconstitution of Mth RNase P activity. (a) Sequence and phylogenetically predicted secondary structure of the Mth RNase P RNA subunit (24). (b) Reconstitution experiment shows Mth Rpp29 is essential for effective Mth RNase P activity against E. coli ptRNA Tyr. The negative control is the substrate alone; positive control is the substrate incubated with E. coli RNase P; Pfu Pop5 was used in place of Mth Pop5.
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We have determined the solution structure of Mth11 (Mth Rpp29), an essential subunit of the RNase P enzyme from the archaebacterium Methanothermobacter thermoautotrophicus (Mth). RNase P is a ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of tRNAs in all three domains of life. In euba...
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Context 1
... in the absence and presence of the four known protein subunits (14). Using conditions similar to those reported for P. horikoshii RNase P reconstitution (15), no single archaeal protein was able to reconstitute measurable activity (data not shown). However, enzyme activity could be obtained by preincubating the RNA with the four proteins ( Fig. 2b). The essentiality of Rpp29 is demonstrated by the lack of activity when Rpp29 was omitted from the reaction (Fig. 2b, compare lanes 5 and ...
Context 2
... for P. horikoshii RNase P reconstitution (15), no single archaeal protein was able to reconstitute measurable activity (data not shown). However, enzyme activity could be obtained by preincubating the RNA with the four proteins ( Fig. 2b). The essentiality of Rpp29 is demonstrated by the lack of activity when Rpp29 was omitted from the reaction (Fig. 2b, compare lanes 5 and ...
Context 3
... P-mediated ptRNA processing. This impasse has now been overcome by the demonstration that a functional archaeal RNase P holoenzyme can be reconstituted from the in vitro-transcribed P RNA and recombinantly ex- pressed and purified Rpp29, Rpp21, Rpp30, and Pop5 proteins of P. horikoshii, a hyperthermophile (15), and Mth, a moderate thermophile (Fig. 2b). Moreover, although the functional roles of the individual protein subunits remain to be elucidated, both studies demonstrate that Rpp29 is essential for enzyme activity in a reconstitution assay. This study on Mth Rpp29 now provides a glimpse into structure-function relationships in archaeal eukaryotic RNase ...
Similar publications
The increased protein proportion of archaeal and eukaryal ribonuclease (RNase) P holoenzymes parallels a vast decrease in
the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus
substantially activate the catalytic (C-) domain of archaeal P RNA from Methanothermobacter, in the a...
Citations
... The five archaeal RPPs (RPP21, RPP29, POP5, RPP30 and L7Ae; see (46) for an exception) are homologous to eukaryotic RPPs (47), allowing archaeal RNase P to serve as a surrogate for its more intractable eukaryotic cousin (25,48,49). Our in vitro reconstitutions of RNase P from different archaea (22,(24)(25)(26) showed that RPP21•RPP29 and POP5•RPP30 work in pairs with the RPR to yield partial RNPs that are catalytically intermediate between the RPR alone and holoenzymes containing all RPPs (22,24,25). ...
The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.
... The large RNase P complex from eukaryotes has been intractable to assembly and biochemical studies in vitro. In contrast, the simpler RNP complex from several archaea has been successfully reconstituted in vitro, providing valuable insights into protein-aided RNA catalysis in this multi-RPP holoenzyme (9)(10)(11)(12)(13)(14)(15)(16). Archaeal RNase P has thus served as an experimental proxy for the eukaryotic enzyme because all archaeal RPPs are orthologous to their cousins in eukaryotic RNase P (1,(17)(18)(19). ...
RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented 'double K-turn' module in type A and type M archaeal RPR variants.
... However, the overall fold is proposed to be similar to the RNA component of the bacterial enzyme based on nucleotide identity and secondary structure conservation, especially in regions pertaining to substrate recognition and enzymatic catalysis. Structures have been solved for each of the five archaeal RNase P protein subunits: RPP21 [94,95], RPP29 [96,97], RPP30 [98], RPP38 [99,100], and POP5 [101] (using the human nomenclature). X-ray crystallography and NMR spectroscopy have both revealed the three-dimensional structure of the RPP21-RPP29 subcomplex [102,103]. ...
Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5’ end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5’ maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field.
... It has also been suggested that this deep evolutionary relationship is evidence of eukaryogenesis from archaea [17]. Given that archaeal RPPs are homologous to some eukaryotic RPPs and that both suites are unrelated to the bacterial one [18,19], archaeal RNase P has been used as a tractable experimental model for biochemical and structural studies designed to uncover the basis for functional dependence of RNase P on multiple RPPs [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. To gain some insight into possible predecessors that eventually led to the higher protein:RNA ratio in archaeal and eukaryotic RNase P, we examined the large number of archaeal genomes now available and used the resulting inventory of archaeal RPPs to analyze the evolution and structure of the protein subunits of archaeal RNase P. ...
... RPP structures fall within common nucleic acid-binding protein families: a zinc ribbon (RPP21), an Sm-like fold (RPP29), an RRM-like fold (POP5), and a TIM barrel (RPP30) [20,21,23,[27][28][29][30][31][32]34]. Moreover, high-resolution structures of the two binary RPP complexes (RPP21•RPP29 and POP5•RPP30) identify the protein-protein interface [23,28,29,45] while high-resolution structures of L7Ae bound to RNA ligands identify the RNA-protein interface [46][47][48][49][50][51][52][53][54][55][56]. ...
... RPP structures fall within common nucleic acid-binding protein families: a zinc ribbon (RPP21), an Sm-like fold (RPP29), an RRM-like fold (POP5), and a TIM barrel (RPP30) [20,21,23,[27][28][29][30][31][32]34]. Moreover, high-resolution structures of the two binary RPP complexes (RPP21‚RPP29 and POP5‚RPP30) identify the protein-protein interface [23,28,29,45] while high-resolution structures of L7Ae bound to RNA ligands identify the RNA-protein interface [46][47][48][49][50][51][52][53][54][55][56]. ...
RNase P, a ribozyme-based ribonucleoprotein (RNP) complex that catalyzes tRNA 5'-maturation, is ubiquitous in all domains of life, but the evolution of its protein components (RNase P proteins, RPPs) is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous) RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs (either alone or as functional binary complexes), we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs.
... Subsequently, four archaeal RPPs (POP5, RPP21, RPP29 and RPP30) were recognized based on homology to these eukaryotic RPPs and experimentally confirmed by their presence in partially purified native Methanothermobacter thermautotrophicus (Mth) RNase P (14). Additional evidence that these RPPs are indeed RNase P subunits came from their ability to aid RPR catalysis in in vitro reconstitution assays of both type A and type M euryarchaeal RNase P (15)(16)(17)(18)(19)(20), which are classified based on the secondary structure of the RPR (21). The majority of euryarchaeal RPR variants are classified as type A [e.g. ...
The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs),
has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the
Mg2+-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific
hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding
interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon
addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides
proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the
binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns,
the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which
L7Ae contributes to RNase P catalysis.
... Leur implication dans l'activité de la RNase P a été confirmée par des tests d'activité de coupure des ARNt prématures après coimmunoprécipitation des protéines (Hall et al., 2002). Plusieurs études ont rapporté la reconstitution in vitro d'une RNase P archée de type A hautement active à partir de la composante ARN et des quatre protéines initialement identifiées (Boomershine et al., 2003;Kouzuma et al., 2003;Terada et al., 2006;Tsai et al., 2006). L'analyse de l'influence des sous-unités protéiques sur l'activité de la RNase P partiellement assemblée montre que les quatre protéines agissent par paire : aPop4-aRpr2 et aPop5-aRpp1 (Tsai et al., 2006), ce qui est cohérent avec les résultats des expériences de double-hybride réalisées sur les protéines de Pyrococcus horikoshii . ...
... La protéine aPop4 se présente sous la forme de six feuillets antiparallèles torsadés entourant un noyau hydrophobe conservé (Boomershine et al., 2003;Sidote et al., 2004). ...
Ribonuclease P (RNase P) is an endoribonuclease that cleaves the 5'-leader sequence of pre-tRNAs. RNase P is conserved between all taxonomic kingdoms and consists of a catalytic RNA subunit and protein components of variable size, from one protein in bacteria to 5 proteins in archae and at least 9 proteins in eukaryotic cells. In addition to RNase P, eukaryotes possess the RNase MRP which has a related RNA core and shares 8 proteins subunits with RNase P but with its own substrate specificity. Here, we propose an original method to purify specifically RNase P and RNase MRP from S. cerevisiae. Using electron microscopy and image processing, we solved the first structure of these two holoenzymes at a resolution of about 1.5 nm. We showed that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules.Structural features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence.
... Archaeal RNase P serves as an experimental alternative to its eukaryotic cousin, 11 which has proven difficult to assemble in vitro despite the availability of constituent subunits in recombinant form, and as a paradigm to uncover the coordination among multiple proteins that aid an RNA catalyst. The latter objective has been assisted by recent advances in functional reconstitution of archaeal RNase P [12][13][14][15][16][17] and elucidation of the high-resolution structures 12,[18][19][20][21][22][23][24][25][26] of the RPPs. Our reconstitution studies revealed that the four archaeal RPPs function as two binary RPP complexes (POP5•RPP30 and RPP21•RPP29), which have large effects on the RPR's catalytic efficiency [e.g., a 4250-fold increase in k cat /K M in Pyrococcus furiosus (Pfu) RNase P]. 17 Subsequent kinetic studies demonstrated a principal role for POP5•RPP30 in enhancing the RPR's rate of pre-tRNA cleavage and RPP21•RPP29 in increasing substrate affinity. ...
... Archaeal RNase P serves as an experimental alternative to its eukaryotic cousin, 11 which has proven difficult to assemble in vitro despite the availability of constituent subunits in recombinant form, and as a paradigm to uncover the coordination among multiple proteins that aid an RNA catalyst. The latter objective has been assisted by recent advances in functional reconstitution of archaeal RNase P [12][13][14][15][16][17] and elucidation of the high-resolution structures 12,[18][19][20][21][22][23][24][25][26] of the RPPs. Our reconstitution studies revealed that the four archaeal RPPs function as two binary RPP complexes (POP5•RPP30 and RPP21•RPP29), which have large effects on the RPR's catalytic efficiency [e.g., a 4250-fold increase in k cat /K M in Pyrococcus furiosus (Pfu) RNase P]. 17 Subsequent kinetic studies demonstrated a principal role for POP5•RPP30 in enhancing the RPR's rate of pre-tRNA cleavage and RPP21•RPP29 in increasing substrate affinity. ...
... 13,16 The RPP structures were solved both individually and as binary complexes. 12,[18][19][20][21][22][23][24][25][26] These structures fall within established nucleic acid binding protein families: an RRM-like fold (POP5), a TIM barrel (RPP30), a zinc ribbon (RPP21) and an Sm-like fold (RPP29). The POP5•RPP30 and RPP21•RPP29 heterodimer structures reveal protein-protein binding interfaces and furnish clues as to possible RNA-binding sites. ...
Ribonuclease P (RNase P) is a ribonucleoprotein complex that utilizes a Mg(2+)-dependent RNA catalyst to cleave the 5' leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg(2+) coordination and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5·RPP30 and RPP21·RPP29). Here, we employed a previously characterized substrate-enzyme conjugate [pre-tRNA(Tyr)-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNA(Tyr)-MjaΔU RPR compared to the wild type, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P toward its functional conformation.
... However, H1 RNA itself can remove the 5′ leader of precursor tRNA in the presence of high concentrations of Mg2+ ions at pH 6 (5). Reconstitution of the full activity of related archaeal RNase P RNPs has been obtained using recombinant Rpp21, Rpp29, Rpp30 and Pop5 proteins (15,23–28). A fifth protein subunit, Rpp38, also known as L7Ae, enhances the optimal reaction temperature of the reconstituted enzyme and increases its catalysis (15,26). ...
Human nuclear RNase P is required for transcription and processing of tRNA. This catalytic RNP has an H1 RNA moiety associated
with ten distinct protein subunits. Five (Rpp20, Rpp21, Rpp25, Rpp29 and Pop5) out of eight of these protein subunits, prepared
in refolded recombinant forms, bind to H1 RNA in vitro. Rpp20 and Rpp25 bind jointly to H1 RNA, even though each protein can interact independently with this transcript. Nuclease
footprinting analysis reveals that Rpp20 and Rpp25 recognize overlapping regions in the P2 and P3 domains of H1 RNA. Rpp21
and Rpp29, which are sufficient for reconstitution of the endonucleolytic activity, bind to separate regions in the catalytic
domain of H1 RNA. Common themes and discrepancies in the RNA-protein interactions between human nuclear RNase P and its related
yeast and archaeal counterparts provide a rationale for the assembly of the fully active form of this enzyme.
... The known archaeal RPPs are homologous to eukaryal proteins Pop5, RPP21, RPP29, RPP30, and RPP38 [21][22][23]. Recombinantly expressed RPPs have been assembled with the in vitro transcribed cognate RPR to reconstitute the holoenzyme from several archaea, including Methanothermobacter thermoautotrophicus (Mth) [24,25] Pyrococcus horikoshii (Pho) [23], Pyrococcus furiosus (Pfu) [26], Methanocaldococcus jannaschii (Mja) [25,27], and Methanococcus maripaludis [22]. Reconstitution studies with Mja, Mth, and Pfu RNase P indicate that four RPPs work in two distinct pairs: Pop5 with RPP30 and RPP21 with RPP29 (designated Pop5-RPP30 and RPP21-RPP29, resp.). ...
... X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR) have been employed to determine the structure of several archaeal RPPs, including RPP29 from Mth [24], Archaeoglobus fulgidus [32], Pho [33], and Pfu [34], Pop5 from Pfu [35] and Pho [36], Pho RPP30 [37], RPP21 from Pho [38] and Pfu [39] and RPP38/L7Ae [40,41]. Structures for the RPP21-RPP29 binary complex were obtained by crystallography for Pho [36] and by NMR for Pfu [34], and of the Pho Pop5-RPP30 pair by crystallography [14]. ...
... Size exclusion chromatography and dynamic light scattering were used to determine the oligomeric state of the Pop5-RPP30 complex ( Figure 2). Free Pfu RPP30 eluted from the gel filtration column with an apparent molecular weight of 24 kDa, matching its monomer mass (24,495 Da). The mixture of RPP30 with an excess of Pop5 yielded two peaks, one corresponding to monomeric Pop5 (13,839 Da) and the other eluting with an apparent mass in excess of that of a heterodimer. ...
RNase P is a highly conserved ribonucleoprotein enzyme that represents a model complex for understanding macromolecular RNA-protein interactions. Archaeal RNase P consists of one RNA and up to five proteins (Pop5, RPP30, RPP21, RPP29, and RPP38/L7Ae). Four of these proteins function in pairs (Pop5-RPP30 and RPP21-RPP29). We have used nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to characterize the interaction between Pop5 and RPP30 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu). NMR backbone resonance assignments of free RPP30 (25 kDa) indicate that the protein is well structured in solution, with a secondary structure matching that observed in a closely related crystal structure. Chemical shift perturbations upon the addition of Pop5 (14 kDa) reveal its binding surface on RPP30. ITC experiments confirm a net 1 : 1 stoichiometry for this tight protein-protein interaction and exhibit complex isotherms, indicative of higher-order binding. Indeed, light scattering and size exclusion chromatography data reveal the complex to exist as a 78 kDa heterotetramer with two copies each of Pop5 and RPP30. These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis.
... In the case of Pyrococcus furiosus RNase P, there was no activation of the RPR when only one Rpp was added and among the six possible two-Rpp combinations, only two were active: Pop5+Rpp30 and Rpp21+Rpp29 (35). Structural and biochemical studies are beginning to furnish insights into the functional coordination between the RPR and Rpps (31,37–42). Footprinting studies reveal that Rpp21–Rpp29 contacts the specificity domain, while Pop5-Rpp30 recognizes the catalytic domain of their cognate RNA subunit (35,43). ...
... These studies collectively demonstrate that the active site rests with the archaeal RPR, an observation also consistent with the fact that none of the recombinant archaeal Rpps (singly or in any combination) show any trace of activity (32,34,35). RNase P holoenzymes from different archaea have been successfully reconstituted using recombinant RPR and Rpps (31,32,34–37). In the case of Pyrococcus furiosus RNase P, there was no activation of the RPR when only one Rpp was added and among the six possible two-Rpp combinations, only two were active: Pop5+Rpp30 and Rpp21+Rpp29 (35). ...
RNase P, a catalytic ribonucleoprotein (RNP), is best known for its role in precursor tRNA processing. Recent discoveries
have revealed that eukaryal RNase P is also required for transcription and processing of select non-coding RNAs, thus enmeshing
RNase P in an intricate network of machineries required for gene expression. Moreover, the RNase P RNA seems to have been
subject to gene duplication, selection and divergence to generate two new catalytic RNPs, RNase MRP and MRP-TERT, which perform
novel functions encompassing cell cycle control and stem cell biology. We present new evidence and perspectives on the functional
diversification of the RNase P RNA to highlight it as a paradigm for the evolutionary plasticity that underlies the extant
broad repertoire of catalytic and unexpected regulatory roles played by RNA-driven RNPs.
