The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Roland K Hartmann - One of the best experts on this subject based on the ideXlab platform.
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structure and mechanistic features of the Prokaryotic minimal RNase P
eLife, 2021Co-Authors: Rebecca Feyh, Roland K Hartmann, Simone Prinz, Nadine B Waeber, Pietro Ivan Giammarinaro, Gert Bange, Georg K A Hochberg, Florian AltegoerAbstract:Endonucleolytic removal of 5'-leader sequences from tRNA Precursor transcriPts (Pre-tRNAs) by ribonuclease P (RNase P) is essential for Protein synthesis. Beyond RNA-based RNase P enzymes, Protein-only versions of the enzyme exert this function in various eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme tyPes belong to distinct subgrouPs of the PIN domain metallonuclease suPerfamily. Homologs of Aquifex RNase P (HARPs) are also exPressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not rePresent the main RNase P activity. Here, we solved the structure of the bacterial HARP from HalorhodosPira haloPhila by cryo-electron microscoPy, revealing a novel screw-like dodecameric assembly. Biochemical exPeriments demonstrate that oligomerization is required for RNase P activity of HARPs. We ProPose that the tRNA substrate binds to an extended sPike-helix (SH) domain that Protrudes from the screw-like assembly to Position the 5'-end in close Proximity to the active site of the neighboring dimer. The structure suggests that eukaryotic PRORPs and Prokaryotic HARPs recognize the same structural elements of Pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for Pre-tRNA Processing by the Prokaryotic HARP system.
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structure and mechanistic features of the Prokaryotic minimal RNase P
bioRxiv, 2021Co-Authors: Rebecca Feyh, Roland K Hartmann, Simone Prinz, Nadine B Waeber, Pietro Ivan Giammarinaro, Gert Bange, Georg K A Hochberg, Florian AltegoerAbstract:Endonucleolytic removal of 59-leader sequences from tRNA Precursor transcriPts (Pre-tRNAs) by RNase P is essential for Protein synthesis. Beyond RNA-based RNase P enzymes, Protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme tyPes belong to distinct subgrouPs of the PIN domain metallonuclease suPerfamily. Homologs of Aquifex RNase P (HARPs) are also exPressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not rePresent the main RNase P activity. Here we solved the structure of the bacterial HARP from HalorhodosPira haloPhila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical exPeriments demonstrate that oligomerization is required for RNase P activity of HARPs. We ProPose that the tRNA substrate binds to an extended sPike-helix (SH) domain that Protrudes from the screw-like assembly to Position the 59-end in close Proximity to the active site of the neighboring dimer subunit. The structure suggests that eukaryotic PRORPs and Prokaryotic HARPs recognize the same structural elements of Pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for Pre-tRNA Processing by the Prokaryotic HARP system.
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homologs of aquifex aeolicus Protein only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei
Iubmb Life, 2019Co-Authors: Thandi S Schwarz, Nadine B. Wäber, Rebecca Feyh, Katrin Weidenbach, Ruth A Schmitz, Anita Marchfelder, Roland K HartmannAbstract:The mature 5'-ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoProtein consisting of a catalytic RNA and one or more Protein subunits. However, in the hyPerthermoPhilic bacterium Aquifex aeolicus and close relatives, RNase P is a Protein-only enzyme consisting of a single tyPe of PolyPePtide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA-based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth PhenotyPes under standard conditions, temPerature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse sPecific tRNA 5'-end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P dePletion strain with comParable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the caPacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth PhenotyPes uPon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA Processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5'-end maturation in archaea, but may well exert a sPecialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109-1116, 2019.
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Minimal and RNA-free RNase P in Aquifex aeolicus.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Astrid I. Nickel, Walter Rossmanith, Nadine B. Wäber, Markus Gößringer, Marcus Lechner, Uwe Linne, Ursula Toth, Roland K HartmannAbstract:RNase P is an essential tRNA-Processing enzyme in all domains of life. We identified an unknown tyPe of Protein-only RNase P in the hyPerthermoPhilic bacterium Aquifex aeolicus: Without an RNA subunit and the smallest of its kind, the 23-kDa PolyPePtide comPrises a metallonuclease domain only. The Protein has RNase P activity in vitro and rescued the growth of Escherichia coli and Saccharomyces cerevisiae strains with inactivations of their more comPlex and larger endogenous ribonucleoProtein RNase P. Homologs of Aquifex RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that A. aeolicus and related Aquificaceae likely acquired HARP by horizontal gene transfer from an archaeon.
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substrate recognition and cleavage site selection by a single subunit Protein only RNase P
Nucleic Acids Research, 2016Co-Authors: Nadia Brillante, Walter Rossmanith, Markus Gosringer, Ursula Toth, Dominik Lindenhofer, Roland K HartmannAbstract:RNase P is the enzyme that removes 5' extensions from tRNA Precursors. With its diversity of enzyme forms-either Protein- or RNA-based, ranging from single PolyPePtides to multi-subunit ribonucleoProteins-the RNase P enzyme family rePresents a unique model system to comPare the evolution of enzymatic mechanisms. Here we Present a comPrehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit Proteinaceous RNase P PRORP3 from ArabidoPsis thaliana. ComPared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger Part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5' or 3' extensions of the tRNA. The cleavage site dePends on the combined dimensions of accePtor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP aPPears mechanistically more similar to the comPlex nuclear ribonucleoProtein enzymes than to the simPler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus aPParently do not necessarily reflect molecular comPosition or evolutionary relationshiP.
Venkat Gopalan - One of the best experts on this subject based on the ideXlab platform.
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Protein cofactors and substrate influence mg2 dePendent structural changes in the catalytic rna of archaeal RNase P
Nucleic Acids Research, 2021Co-Authors: Ila A Marathe, Stella M Lai, Vicki H Wysocki, Walter J Zahurancik, Michael G Poirier, Venkat GopalanAbstract: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.
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the rice RNase P Protein subunit rPP30 confers broad sPectrum resistance to fungal and bacterial Pathogens
Plant Biotechnology Journal, 2021Co-Authors: Yehui Xiong, Venkat Gopalan, Lien B Lai, Kai Zhang, Houxiang Kang, Liangying Dai, Guoliang Wang, Wende LiuAbstract:RNase P functions either as a catalytic ribonucleoProtein (RNP) or as an RNA-free PolyPePtide to catalyse RNA Processing, Primarily tRNA 5' maturation. To the growing evidence of non-canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P RPP30 in innate immunity. This Protein (encoded by LOC_Os11g01074) was uncovered as the toP hit in yeast two-hybrid assays Performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRPP30 are localized to the rice nucleus, OsRPP30 exPression increased Post-infection by Pyricularia oryzae (syn. MagnaPorthe oryzae), and OsRPP30 deacetylation coincided with HDT701 overexPression in vivo. OverexPression of OsRPP30 in transgenic rice increased exPression of defence genes and generation of reactive oxygen sPecies after Pathogen-associated molecular Pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae Pv. oryzae) Pathogen. Knockout of OsRPP30 yielded the oPPosite PhenotyPes. Moreover, HA-tagged OsRPP30 co-Purified with RNase P Pre-tRNA cleavage activity. Interestingly, OsRPP30 is conserved in grass croPs, including a near-identical C-terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRPP30 Plays an imPortant role in rice immune resPonse to Pathogens and Provides a new aPProach to generate broad-sPectrum disease-resistant rice cultivars.
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biogenesis of RNase P rna from an intron requires co assembly with cognate Protein subunits
Nucleic Acids Research, 2019Co-Authors: Geeta Palsule, Venkat Gopalan, Amanda SimcoxAbstract:RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoProtein, removes the 5' leader from Precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcriPt generated with mature termini. In the branch of the arthroPod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcriPt and requires Processing from intron sequences for maturation. We demonstrate here that the DrosoPhila intronic-RPR Precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5') and the RNA exosome (3'). Processing is regulated by a subset of RNase P Proteins (RPPs) that Protects the nascent RPR from degradation, the tyPical fate of excised introns. Our results indicate that the biogenesis of RPR in vivo entails interaction of RPPs with the nascent RNA to form the RNase P holoenzyme and suggests that a new Pathway arose in arthroPods by cooPting ancient mechanisms common to Processing of other noncoding RNAs.
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both kinds of RNase P in all domains of life surPrises galore
RNA, 2019Co-Authors: Charles J Daniels, Lien B Lai, Tienhao Chen, Venkat GopalanAbstract:RNase P, an essential housekeePing endonuclease needed for 5'-Processing of tRNAs, exists in two distinct forms: one with an RNA- and the other with a Protein-based active site. The notion that the Protein form of RNase P exists only in eukaryotes has been uPended by the recent discovery of a Protein-only variant in Bacteria and Archaea. The use of these two divergent scaffolds, shaPed by convergent evolution, in all three domains of life insPires questions relating to the ancestral form of RNase P, as well as their origins and function(s) in vivo. Results from our analysis of Publicly available bacterial and archaeal genomes suggest that the widesPread RNA-based ribonucleoProtein variant is likely the ancient form. We also discuss the Possible genetic origins and function of RNase P, including how the simultaneous Presence of its variants may contribute to the fitness of their host organisms.
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chance and necessity in the evolution of RNase P
RNA, 2018Co-Authors: Venkat Gopalan, Nayef Jarrous, Andrey S KrasilnikovAbstract:RNase P catalyzes 5'-maturation of tRNAs in all three domains of life. This Primary function is accomPlished by either a ribozyme-centered ribonucleoProtein (RNP) or a Protein-only variant (with one to three PolyPePtides). The large, multicomPonent archaeal and eukaryotic RNase P RNPs aPPear disProPortionate to the simPlicity of their role in tRNA 5'-maturation, PromPting the question of why the seemingly gratuitously comPlex RNP forms of RNase P were not rePlaced with simPler Protein counterParts. Here, motivated by growing evidence, we consider the hyPothesis that the large RNase P RNP was retained as a direct consequence of multiPle roles Played by its comPonents in Processes that are not related to the canonical RNase P function.
David R Engelke - One of the best experts on this subject based on the ideXlab platform.
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RNase P enzymes divergent scaffolds for a conserved biological reaction
RNA Biology, 2013Co-Authors: Michael J Howard, Carol A Fierke, Xin Liu, Bradley P Klemm, Wan Hsin Lim, Markos Koutmos, David R EngelkeAbstract:Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of Precursor-tRNAs (Pre-tRNA) and is conserved in all domains of life. However, the comPosition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 Proteins. Recently, a Protein-only form of RNase P was discovered in mitochondria and chloroPlasts of many higher eukaryotes. This Proteinaceous RNase P (PRORP) rePresents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from ArabidoPsis thaliana and sPeculate on the reasons for the rePlacement of catalytic RNA by a Protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoProtein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the rePlacement of the RNA with a Protein catalyst. The emergence of a...
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accumulation of noncoding rna due to an RNase P defect in saccharomyces cerevisiae
RNA, 2011Co-Authors: Michael C Marvin, Scott C. Walker, Sandra Claudermunster, Ali Sarkeshik, John R Yates, Lars M Steinmetz, David R EngelkeAbstract:Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of the 5' leader of Pre-tRNAs. In addition, a growing number of non-tRNA substrates have been identified in various organisms. RNase P varies in comPosition, as bacterial RNase P contains a catalytic RNA core and one Protein subunit, while eukaryotic nuclear RNase P retains the catalytic RNA but has at least nine Protein subunits. The additional eukaryotic Protein subunits most likely Provide additional functionality to RNase P, with one Possibility being additional RNA recognition caPabilities. To investigate the Possible range of additional RNase P substrates in vivo, a strand-sPecific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting that nuclear RNase P ParticiPates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcriPts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either comPromised sPlicing or failure to efficiently turn over Pre-mRNAs that do not enter the sPlicing Pathway. Taken together with the high comPlexity of the nuclear RNase P holoenzyme and its relatively nonsPecific caPacity to bind and cleave mixed sequence RNAs, these data suggest that nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in Pre-tRNA biogenesis.
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binding and cleavage of unstructured rna by nuclear RNase P
RNA, 2011Co-Authors: Michael C Marvin, Scott C. Walker, Carol A Fierke, David R EngelkeAbstract:Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is Processing the 5′ leader of Pre-tRNAs. ComPared to bacterial RNase P, which contains a single small Protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more comPlex, containing nine Proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to Possess unique RNA binding caPabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dePendent manner, suggesting that multiPle interactions are required for high affinity binding. Mixed-sequence RNAs from Protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to Poly(U) homoPolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiPle Preferential cleavage sites that do not corresPond to identifiable consensus structures or sequences. In addition, Pre-tRNATyr, Poly(U)50 RNA, and mixed-sequence RNA cross-link with Purified RNase P in the RNA subunit RPr1 near the active site in “Conserved Region I,” although the exact Positions vary. Additional contacts between Poly(U)50 and the RNase P Proteins RPr2P and PoP4P were identified. We conclude that unstructured RNAs interact with multiPle Protein and RNA contacts near the RNase P RNA active site, but that cleavage dePends on the nature of interaction with the active site.
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eukaryote RNase P and RNase mrP
2010Co-Authors: Scott C. Walker, Michael C Marvin, David R EngelkeAbstract:Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the cleavage of the 5′ leader sequence from Precursor tRNAs (Pre-tRNAs). Most forms of RNase P are ribonucleoProteins and the bacterial enzyme Possesses a single catalytic RNA and one small Protein. In eukaryotes, the enzyme retains a structurally related, catalytic RNA subunit but has a vastly increased Protein comPosition with at least nine Protein subunits in yeast and at least ten in humans. The reasons for this additional Protein comPlexity over the bacterial and archaeal RNase P enzymes are not currently understood and Potential roles including the acquisition of additional substrates are discussed. Furthermore, in the eukaryote RNase P has evolved into a distinct but closely related enzyme, RNase MRP. This Paralogous enzyme has a structurally related RNA subunit and ten Protein subunits in yeast, eight of which are also found in the RNase P enzyme. RNase MRP has distinct substrate sPecificities, Primarily involved in ribosomal RNA biogenesis, but also cleaving mitochondrial RNA and mRNAs involved in cell cycle control. We review current information regarding the nuclear RNase P and RNase MRP enzymes in the eukaryotes, focusing on the relationshiP between these enzymes by examining their comPosition, structure and substrates.
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Pre trna turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by Product release
RNA, 2009Co-Authors: John Hsieh, Scott C. Walker, Carol A Fierke, David R EngelkeAbstract:Ribonuclease P (RNase P) is a ribonucleoProtein that catalyzes the 59 maturation of Precursor transfer RNA in the Presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA comPonent and a single essential but catalytically inactive Protein. In contrast, yeast nuclear RNase P is more comPlex with one RNA subunit and nine Protein subunits. We have devised an affinity Purification Protocol to gently and raPidly Purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In Pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amPlitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting steP in turnover occurs after Pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dePendence on temPerature that results in nonlinear Arrhenius Plots, suggesting that a kinetically imPortant conformational change occurs during catalysis. Finally, deletion of the 39 trailer in Pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, desPite marked differences in subunit comPosition, the minimal kinetic mechanism for cleavage of PretRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.
Norman R Pace - One of the best experts on this subject based on the ideXlab platform.
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solution structure of RNase P rna
RNA, 2011Co-Authors: Alexei V Kazantsev, Robert P Rambo, Sina Karimpour, John Santalucia, John A Tainer, Norman R PaceAbstract:The ribonucleoProtein enzyme ribonuclease P (RNase P) Processes tRNAs by cleavage of Precursor-tRNAs. RNase P is a ribozyme: The RNA comPonent catalyzes tRNA maturation in vitro without Proteins. Remarkable features of RNase P include multiPle turnovers in vivo and ability to Process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary comPlex with substrate, have been determined by X-ray crystallograPhy. However, crystal structures of free RNA are significantly different from the ternary comPlex, and the solution structure of the RNA is unknown. Here, we rePort solution structures of three Phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermoPhilus, determined using small angle X-ray scattering (SAXS) and selective 29-hydroxyl acylation analyzed by Primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the emPirical data of these RNAs in solution under the high ionic strength required for catalytic activity.
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maPPing metal binding sites in the catalytic domain of bacterial RNase P rna
RNA, 2009Co-Authors: Alexei V Kazantsev, A A Krivenko, Norman R PaceAbstract:Ribonuclease P (RNase P) is a ribonucleoProtein enzyme that contains a universally conserved, catalytically active RNA comPonent. RNase P RNA requires divalent metal ions for folding, substrate binding, and catalysis. DesPite recent advances in understanding the structure of RNase P RNA, no comPrehensive analysis of metal-binding sites has been rePorted, in Part due to the Poor crystallization ProPerties of this large RNA. We have develoPed an abbreviated yet still catalytic construct, Bst P7Δ RNA, which contains the catalytic domain of the bacterial RNase P RNA and has imProved crystallization ProPerties. We use this mutant RNA as well as the native RNA to maP metal-binding sites in the catalytic core of the bacterial RNase P RNA, by anomalous scattering in diffraction analysis. The results Provide insight into the interPlay between RNA structure and focalization of metal ions, and a structural basis for some Previous biochemical observations with RNase P. We use electrostatic calculations to extract the Potential functional significance of these metal-binding sites with resPect to binding Mg2+. The results suggest that with at least one imPortant excePtion of sPecific binding, these sites mainly maP areas of diffuse association of magnesium ions.
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bacterial RNase P a new view of an ancient enzyme
Nature Reviews Microbiology, 2006Co-Authors: Alexei V Kazantsev, Norman R PaceAbstract:Ribonuclease P (RNase P) is a ubiquitous endonuclease that catalyses the maturation of the 5' end of transfer RNA (tRNA). Although it carries out a biochemically simPle reaction, RNase P is a comPlex ribonucleoProtein Particle comPosed of a single large RNA and at least one Protein comPonent. In bacteria and some archaea, the RNA comPonent of RNase P can catalyse tRNA maturation in vitro in the absence of Proteins. The discovery of the catalytic activity of the bacterial RNase P RNA triggered numerous mechanistic and biochemical studies of the reactions catalysed by the RNA alone and by the holoenzyme and, in recent years, structures of individual comPonents of the RNase P holoenzyme have been determined. The goal of the Present review is to summarize what is known about the bacterial RNase P, and to bring together the recent structural results with extensive earlier biochemical and Phylogenetic findings.
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RNase P interface of the rna and Protein worlds
Trends in Biochemical Sciences, 2006Co-Authors: Donald Evans, Steven M Marquez, Norman R PaceAbstract:Ribonuclease P (RNase P) is an endonuclease involved in Processing tRNA. It contains both RNA and Protein subunits and occurs in all three domains of life: namely, Archaea, Bacteria and Eukarya. The RNase P RNA subunits from bacteria and some archaea are catalytically active in vitro, whereas those from eukaryotes and most archaea require Protein subunits for activity. RNase P has been characterized biochemically and genetically in several systems, and detailed structural information is emerging for both RNA and Protein subunits from Phylogenetically diverse organisms. In vitro reconstitution of activity is Providing insight into the role of Proteins in the RNase P holoenzyme. Together, these findings are beginning to imPart an understanding of the coevolution of the RNA and Protein worlds.
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structural PersPective on the activation of RNase P rna by Protein
Nature Structural & Molecular Biology, 2005Co-Authors: Amy H Buck, Alexei V Kazantsev, Andrew B Dalby, Norman R PaceAbstract:RibonucleoProtein Particles are central to numerous cellular Pathways, but their study in vitro is often comPlicated by heterogeneity and aggregation. We describe a new technique to characterize these comPlexes traPPed as homogeneous sPecies in a nondenaturing gel. Using this technique, in conjunction with PhosPhorothioate footPrinting analysis, we identify the Protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a Protein cofactor to catalyze the 5′ maturation of Precursor transfer RNA (Pre-tRNA). Our results show that the Protein binds to a Patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the Protein in substrate recognition and suPPort a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.
Carol A Fierke - One of the best experts on this subject based on the ideXlab platform.
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kinetic mechanism of human mitochondrial RNase P
bioRxiv, 2019Co-Authors: Xin Liu, Aranganathan Shanmuganathan, Bradley P Klemm, Michael J Howard, Wan Hsin Lim, Markos Koutmos, Carol A FierkeAbstract:A first steP in Processing mitochondrial Precursor tRNA (Pre-tRNA) is cleavage of the 5-Prime leader catalyzed by ribonuclease P (RNase P). Human mitochondrial RNase P (mtRNase P) is comPosed of three Protein subunits: mitochondrial RNase P Protein (MRPP) 1, 2 and 3. Even though MRPP3 is the metallonuclease subunit resPonsible for catalysis, cleavage is observed only in the Presence of the MRPP1/2 subcomPlex. To understand the functional role of MRPP1/2, we reconstituted human mitochondrial RNase P in vitro and Performed kinetic and thermodynamic analyses. MRPP1/2 significantly enhances both the catalytic activity and the aPParent substrate affinity of mtRNase P. Additionally, Pull-down and binding data demonstrate synergy between binding Pre-tRNA and formation of a catalytically active MRPP1/2/3 comPlex. These data suggest that conformational changes in the MRPP1/2-Pre-tRNA comPlex lead to Protein-Protein or Protein-RNA interactions that increase both MRPP3 recognition and cleavage efficiency. This work Presents the first kinetic model for human mtRNase P, Providing a fundamental framework for the function of MRPP1/2 for recognition and Processing of Pre-tRNA.
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fluorescence based real time activity assays to identify RNase P inhibitors
Methods of Molecular Biology, 2017Co-Authors: Yu Chen, Xin Liu, Carol A FierkeAbstract:Transfer RNA is transcribed as Precursor molecules that are Processed before ParticiPating in translation catalyzed by the ribosome. Ribonuclease P is the endonuclease that catalyzes the 5' end maturation of Precursor tRNA and it is essential for cell survival. Bacterial RNase P has a distinct subunit comPosition comPared to the eukaryal counterParts; therefore, it is an attractive antibacterial target. Here, we describe a real-time fluorescence-based RNase P activity assay using fluorescence Polarization/anisotroPy with a 5' end fluorescein-labeled Pre-tRNAAsP substrate. This FP/FA assay is sensitive, robust, and easy to transition to a high-throughPut mode and it also detects ligands that interact with Pre-tRNA. We aPPly this FP/FA assay to measure Bacillus subtilis RNase P activity under single and multiPle turnover conditions in a continuous format and a high-throughPut screen of inhibitors, as well as determining the dissociation constant of Pre-tRNA for small molecules.
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RNase P enzymes divergent scaffolds for a conserved biological reaction
RNA Biology, 2013Co-Authors: Michael J Howard, Carol A Fierke, Xin Liu, Bradley P Klemm, Wan Hsin Lim, Markos Koutmos, David R EngelkeAbstract:Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of Precursor-tRNAs (Pre-tRNA) and is conserved in all domains of life. However, the comPosition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 Proteins. Recently, a Protein-only form of RNase P was discovered in mitochondria and chloroPlasts of many higher eukaryotes. This Proteinaceous RNase P (PRORP) rePresents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from ArabidoPsis thaliana and sPeculate on the reasons for the rePlacement of catalytic RNA by a Protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoProtein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the rePlacement of the RNA with a Protein catalyst. The emergence of a...
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the rnr motif of b subtilis RNase P Protein interacts with both Prna and Pre trna to stabilize an active conformer
RNA, 2011Co-Authors: Kristin S Koutmou, Jeremy J Daystorms, Carol A FierkeAbstract:Ribonuclease P (RNase P) catalyzes the metal-dePendent 5′ end maturation of Precursor tRNAs (Pre-tRNAs). In Bacteria, RNase P is comPosed of a catalytic RNA (PRNA) and a Protein subunit (P Protein) necessary for function in vivo. The P Protein enhances Pre-tRNA affinity, selectivity, and cleavage efficiency, as well as modulates the cation requirement for RNase P function. Bacterial P Proteins share little sequence conservation although the Protein structures are homologous. Here we combine site-directed mutagenesis, affinity measurements, and single turnover kinetics to demonstrate that two residues (R60 and R62) in the most highly conserved region of the P Protein, the RNR motif (R60–R68 in Bacillus subtilis), stabilize PRNA comPlexes with both P Protein (PRNA•P Protein) and Pre-tRNA (PRNA•P Protein•Pre-tRNA). Additionally, these data indicate that the RNR motif enhances a metal-stabilized conformational change in RNase P that accomPanies substrate binding and is essential for efficient catalysis. Stabilization of this conformational change contributes to both the decreased metal requirement and the enhanced substrate recognition of the RNase P holoenzyme, illuminating the role of the most highly conserved region of P Protein in the RNase P reaction Pathway.
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binding and cleavage of unstructured rna by nuclear RNase P
RNA, 2011Co-Authors: Michael C Marvin, Scott C. Walker, Carol A Fierke, David R EngelkeAbstract:Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is Processing the 5′ leader of Pre-tRNAs. ComPared to bacterial RNase P, which contains a single small Protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more comPlex, containing nine Proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to Possess unique RNA binding caPabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dePendent manner, suggesting that multiPle interactions are required for high affinity binding. Mixed-sequence RNAs from Protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to Poly(U) homoPolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiPle Preferential cleavage sites that do not corresPond to identifiable consensus structures or sequences. In addition, Pre-tRNATyr, Poly(U)50 RNA, and mixed-sequence RNA cross-link with Purified RNase P in the RNA subunit RPr1 near the active site in “Conserved Region I,” although the exact Positions vary. Additional contacts between Poly(U)50 and the RNase P Proteins RPr2P and PoP4P were identified. We conclude that unstructured RNAs interact with multiPle Protein and RNA contacts near the RNase P RNA active site, but that cleavage dePends on the nature of interaction with the active site.
