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Carol A. Fierke - One of the best experts on this subject based on the ideXlab platform.
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The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions.
Biomolecules, 2016Co-Authors: Bradley P. Klemm, Yu Chen, Xin Liu, Kipchumba J. Kaitany, Michael J. Howard, Carol A. FierkeAbstract: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.
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imPortance of rna Protein interactions in bacterial Ribonuclease P structure and catalysis
Biopolymers, 2007Co-Authors: Kristin J Smith, John Hsieh, Carol A. FierkeAbstract:Ribonuclease P (RNase P) is a ribonucleoProtein (RNP) comPlex that catalyzes the metal-dePendent maturation of the 5 0 end of Precursor tRNAs (Pre-tRNAs) in all organisms. RNase P is comPrised of a catalytic RNA (P RNA), and at least one essential Protein (P Protein). Although P RNA is the catalytic subunit of the enzyme and is active in the absence of P Protein under high salt concentrations in vitro, the Protein is still required for enzyme activity in vivo. Therefore, the function of the P Protein and how it interacts with both P RNA and Pre- tRNA have been the focus of much ongoing research. RNA-Protein interactions in RNase P serve a number of critical roles in the RNP including stabilizing the structure, and enhancing the affinity for substrates and metal ions. This review examines the role of RNA-Protein interactions in bacterial RNase P from both structural and mechanistic PersPectives. # 2007 Wiley Periodicals,
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ImPortance of RNA-Protein interactions in bacterial Ribonuclease P structure and catalysis
Biopolymers, 2007Co-Authors: J. Kristin Smith, John Hsieh, Carol A. FierkeAbstract:Ribonuclease P (RNase P) is a ribonucleoProtein (RNP) comPlex that catalyzes the metal-dePendent maturation of the 5' end of Precursor tRNAs (Pre-tRNAs) in all organisms. RNase P is comPrised of a catalytic RNA (P RNA), and at least one essential Protein (P Protein). Although P RNA is the catalytic subunit of the enzyme and is active in the absence of P Protein under high salt concentrations in vitro, the Protein is still required for enzyme activity in vivo. Therefore, the function of the P Protein and how it interacts with both P RNA and Pre-tRNA have been the focus of much ongoing research. RNA-Protein interactions in RNase P serve a number of critical roles in the RNP including stabilizing the structure, and enhancing the affinity for substrates and metal ions. This review examines the role of RNA-Protein interactions in bacterial RNase P from both structural and mechanistic PersPectives.
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Roles of Protein subunits in RNA-Protein comPlexes: lessons from Ribonuclease P.
Biopolymers, 2003Co-Authors: John Hsieh, Andrew J. Andrews, Carol A. FierkeAbstract:RibonucleoProteins (RNP) are involved in many essential Processes in life. However, the roles of RNA and Protein subunits in an RNP comPlex are often hard to dissect. In many RNP comPlexes, including the ribosome and the GrouP II introns, one main function of the Protein subunits is to facilitate RNA folding. However, in other systems, the Protein subunits may Perform additional functions, and can affect the biological activities of the RNP comPlexes. In this review, we use Ribonuclease P (RNase P) as an examPle to illustrate how the Protein subunit of this RNP affects different asPects of catalysis. RNase P Plays an essential role in the Processing of the Precursor to transfer RNA (Pre-tRNA) and is found in all three domains of life. While every cell has an RNase P (Ribonuclease P) enzyme, only the bacterial and some of the archaeal RNase P RNAs (RNA comPonent of RNase P) are active in vitro in the absence of the RNase P Protein. RNase P is a remarkable enzyme in the fact that it has a conserved catalytic core comPosed of RNA around which a diverse array of Protein(s) interact to create the RNase P holoenzyme. This combination of highly conserved RNA and altered Protein comPonents is a Puzzle that allows the dissection of the functional roles of Protein subunits in these RNP comPlexes.
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sPecific PhosPhorothioate substitutions Probe the active site of bacillus subtilis Ribonuclease P
RNA, 2002Co-Authors: Sharon M. Crary, Jeffrey C. Kurz, Carol A. FierkeAbstract:Ribonuclease P (RNase P) is a ribonucleoProtein that requires magnesium ions to catalyze the 5' maturation of transfer RNA. To identify interactions essential for catalysis, the ProPerties of RNase P containing single sulfur substitutions for nonbridging PhosPhodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic exPeriments. Sulfur substitution at the nonbridging oxygens of the PhosPhodiester bond of nucleotide U51 only modestly affects catalysis. However, PhosPhorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300-4,000-fold for P RNA and 500-15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of Pre-tRNA(AsP), highlighting the imPortance of this region for catalysis. Furthermore, addition of manganese enhances Pre-tRNA cleavage catalyzed by B. subtilis RNase P RNA containing an SP PhosPhorothioate modification at A49, as observed for Escherichia coli P RNA [Christian et al., RNA, 2000, 6:511-519], suggesting that an essential metal ion may be coordinated at this site. In contrast, no manganese rescue is observed for the A49 SP PhosPhorothioate modification in RNase P holoenzyme. These differential manganese rescue effects, along with affinity cleavage, suggest that the Protein comPonent may interact with a metal ion bound near A49 in helix P4 of P RNA.
Michael E. Harris - One of the best experts on this subject based on the ideXlab platform.
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Determination of the SPecificity LandscaPe for Ribonuclease P Processing of Precursor tRNA 5′ Leader Sequences
ACS chemical biology, 2016Co-Authors: Courtney N. Niland, Jing Zhao, Hsuan Chun Lin, David R. Anderson, Eckhard Jankowsky, Michael E. HarrisAbstract:Maturation of tRNA dePends on a single endonuclease, Ribonuclease P (RNase P), to remove highly variable 5' leader sequences from Precursor tRNA transcriPts. Here, we use high-throughPut enzymology to rePort multiPle-turnover and single-turnover kinetics for Escherichia coli RNase P Processing of all Possible 5' leader sequences, including nucleotides contacting both the RNA and Protein subunits of RNase P. The results reveal that the identity of N(-2) and N(-3) relative to the cleavage site at N(1) Primarily control alternative substrate selection and act at the level of association not the cleavage steP. As a consequence, the sPecificity for N(-1), which contacts the active site and contributes to catalysis, is suPPressed. This study demonstrates high-throughPut RNA enzymology as a means to globally determine RNA sPecificity landscaPes and reveals the mechanism of substrate discrimination by a widesPread and essential RNA-Processing enzyme.
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determination of the sPecificity landscaPe for Ribonuclease P Processing of Precursor trna 5 leader sequences
ACS Chemical Biology, 2016Co-Authors: Courtney N. Niland, Jing Zhao, Hsuan Chun Lin, David R. Anderson, Eckhard Jankowsky, Michael E. HarrisAbstract:Maturation of tRNA dePends on a single endonuclease, Ribonuclease P (RNase P), to remove highly variable 5′ leader sequences from Precursor tRNA transcriPts. Here, we use high-throughPut enzymology to rePort multiPle-turnover and single-turnover kinetics for Escherichia coli RNase P Processing of all Possible 5′ leader sequences, including nucleotides contacting both the RNA and Protein subunits of RNase P. The results reveal that the identity of N(−2) and N(−3) relative to the cleavage site at N(1) Primarily control alternative substrate selection and act at the level of association not the cleavage steP. As a consequence, the sPecificity for N(−1), which contacts the active site and contributes to catalysis, is suPPressed. This study demonstrates high-throughPut RNA enzymology as a means to globally determine RNA sPecificity landscaPes and reveals the mechanism of substrate discrimination by a widesPread and essential RNA-Processing enzyme.
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Recent insights into the structure and function of the ribonucleoProtein enzyme Ribonuclease P.
Current opinion in structural biology, 2003Co-Authors: Michael E. Harris, Eric L. ChristianAbstract:In bacteria, the tRNA-Processing endonuclease Ribonuclease P is comPosed of a large (∼400 nucleotide) catalytic RNA and a smaller (∼100 amino acid) Protein subunit that is essential for substrate recognition. Current biochemical and bioPhysical investigations are Providing fresh insights into the modular architecture of the ribozyme, the mechanisms of substrate sPecificity and the role of essential metal ions in catalysis. Together with recent high-resolution structures of Portions of the ribozyme, these findings are beginning to reveal how the functions of RNA and Protein are coordinated in this ribonucleoProtein enzyme.
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Structure and Catalytic Function of the Bacterial Ribonuclease P Ribozyme
Cold Spring Harbor Monograph Archive, 1998Co-Authors: Michael E. Harris, Daniel N. Frank, Norman R. PaceAbstract:Ribonuclease P (RNase P) was Present in the common ancestor of all life and was comPosed, at least in Part, of RNA. This PersPective is based on the observation that RNase P activity is Present in organisms belonging to all three modern Phylogenetic domains, and all of the cellular enzymes resPonsible for that activity have essential RNA subunits (see, e.g., Guerrier-Takada et al. 1983; Cherayil et al. 1987; LaGrandeur et al. 1993). ComParison of RNase P RNA sequences from rePresentatives of all three Phylogenetic domains reveals clear sequence and structural similarities (see, e.g., Forster and Altman 1990b; Tranguch and Engelke 1993; Haas et al. 1996a; Chen and Pace 1997). Thus, bacterial, archaeal and eukaryal RNase P RNAs are “homologous,” that is, they share ancestry. Such ancient heritage indicates a central role for RNA in the function of this enzyme. RNase P catalyzes the hydrolysis of a sPecific PhosPhodiester bond in Pre-tRNA to generate the 5′ end of mature tRNA. Understanding the role that the RNA Plays in the function of RNase P requires knowledge of the enzyme’s structure and catalytic ProPerties. In bacteria, the RNase P holoenzyme is a heterodimer comPosed of a single Protein (~120 amino acids) and RNA (~400 nucleotides) comPonents. In vivo, both subunits are essential for Pre-tRNA Processing and cell viability (Schedl and Primakoff 1973). However, under in vitro reaction conditions of high ionic strength, bacterial RNase P RNAs retain the substrate-binding and catalytic ProPerties of the holoenzyme, and so are one class of ribozymes (Guerrier-Takada...
Makoto Kimura - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for activation of an archaeal Ribonuclease P RNA by Protein cofactors.
Bioscience biotechnology and biochemistry, 2017Co-Authors: Makoto KimuraAbstract:Ribonuclease P (RNase P) is an endoRibonuclease that catalyzes the Processing of the 5'-leader sequence of Precursor tRNA (Pre-tRNA) in all Phylogenetic domains. We have found that RNase P in the hyPerthermoPhilic archaeon Pyrococcus horikoshii OT3 consists of RNase P RNA (PhoPRNA) and five Protein cofactors designated PhoPoP5, PhoRPP21, PhoRPP29, PhoRPP30, and PhoRPP38. Biochemical characterizations over the Past 10 years have revealed that PhoPoP5 and PhoRPP30 fold into a heterotetramer and cooPerate to activate a catalytic domain (C-domain) in PhoPRNA, whereas PhoRPP21 and PhoRPP29 form a heterodimer and function together to activate a sPecificity domain (S-domain) in PhoPRNA. PhoRPP38 Plays a role in elevation of the oPtimum temPerature of RNase P activity, binding to kink-turn (K-turn) motifs in two stem-looPs in PhoPRNA. This review describes the structural and functional information on P. horikoshii RNase P, focusing on the structural basis for the PhoPRNA activation by the five RNase P Proteins.
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Archaeal Ribonuclease P Proteins have Potential for biotechnological aPPlications where Precise hybridization of nucleic acids is needed
Bioscience biotechnology and biochemistry, 2015Co-Authors: Mitsuru Miyanoshita, Yoshimitsu Kakuta, Takashi Nakashima, Makoto KimuraAbstract:Fluorescence resonance energy transfer-based assay showed that archaeal Ribonuclease P (RNase P) Proteins significantly Promoted DNA annealing and strand disPlacement. Moreover, we found that archaeal RNase P Proteins could discriminate nucleotide exchanges in DNA chains via their activity accelerating DNA strand disPlacement, suggesting that they have Potential for biotechnological aPPlication to genetic diagnosis.
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A distinct binding mode of archaeal Ribonuclease P Proteins to RNA.
Bioscience biotechnology and biochemistry, 2012Co-Authors: Masato Ishihara, Etsuko Nishimoto, Shoji Yamashita, Yoshimitsu Kakuta, Makoto KimuraAbstract:The Ribonuclease P (RNase P) in the hyPerthermoPhilic archaeon Pyrococcus horikoshii comPrises RNA (PhoPRNA) and five Proteins. We analyzed the RNA binding mode of the Protein, using a Pair of comPlementary fluorescence-labeled oligoribonucleotides. Fluorescence resonance energy transfer (FRET)-based assays suggested that the RNase P Proteins assist PhoPRNA in attaining a functionally active conformation via a distinct mode of binding.
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Structural Biology of the Ribonuclease P in the HyPerthermoPhilic Archaeon Pyrococcus horikoshii OT3
Microorganisms in Sustainable Agriculture and Biotechnology, 2011Co-Authors: Makoto Kimura, Yoshimitsu KakutaAbstract:Pyrococcus horikoshii OT3 is a hyPerthermoPhilic archaeaon isolated from hydrothermal fluid. Because of their genetic features as well as hyPerthermoPhilic ProPerties, macromolecules Produced by this thermoPhilic bacterium have served as an excellent model for structural biology. Ribonuclease P (RNase P) is a ribonucleoProtein comPlex involved in the Processing of the 5′-leader sequence of Precursor tRNA (Pre-tRNA). We found that RNase P RNA (PhoPRNA) and five Proteins in P. horikoshii OT3 reconstituted RNase P activity that exhibited enzymatic ProPerties like those of the authentic enzyme. A mutational analysis indicated that nucleotides A40, A41, and U44 in PhoPRNA are crucial for catalysis, strongly suggesting that PhoPRNA catalyzes the hydrolysis of Pre-tRNA in aPProximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the Proteins. The P. horikoshii RNase P Proteins are Predominantly involved in oPtimization of PhoPRNA’s conformation, though individually they are disPensable for RNase P activity in vitro. This chaPter summarizes structure-function relationshiPs of the P. horikoshii RNase P subunits, including the high-resolution structural information that is currently available for the Protein subunits.
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Crystal structure of a Ribonuclease P Protein Ph1601P from Pyrococcus horikoshii OT3: an archaeal homologue of human nuclear Ribonuclease P Protein RPP21.
Biochemistry, 2005Co-Authors: Yoshimitsu Kakuta, Tomoyuki Numata, Ikuko Ishimatsu, Isao Tanaka, Kazumi Kimura, Min Yao, Makoto KimuraAbstract:Ribonuclease P (RNase P) is a ribonucleoProtein comPlex involved in the removal of 5' leader sequences from tRNA Precursors (Pre-tRNA). The human Protein RPP21 is essential for human RNase P activity in tRNA Processing in vitro. The crystal structure of Ph1601P from the hyPerthermoPhilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of RPP21, was determined using the multiPle anomalous disPersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 A resolution. Ph1601P comPrises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaPed structure. The N-terminal domain consists of two long alPha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic Potential rePresentation indicates the Presence of Positively charged clusters along the L arms, suggesting a Possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and aPPears to stabilize the relative Positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the Protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central looP and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr44, Arg65, and Arg84 Play a modest role in the activity.
Norman R. Pace - One of the best experts on this subject based on the ideXlab platform.
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Structure and function of eukaryotic Ribonuclease P RNA.
Molecular cell, 2006Co-Authors: Steven M. Marquez, Julian J.-l. Chen, Donald Evans, Norman R. PaceAbstract:Ribonuclease P (RNase P) is the ribonucleoProtein endonuclease that Processes the 5' ends of Precursor tRNAs. Bacterial and eukaryal RNase P RNAs had the same Primordial ancestor; however, they were molded differently by evolution. RNase P RNAs of eukaryotes, in contrast to bacterial RNAs, are not catalytically active in vitro without Proteins. By comParing the bacterial and eukaryal RNAs, we can begin to understand the transitions made between the RNA and Protein-dominated worlds. We rePort, based on crosslinking studies, that eukaryal RNAs, although catalytically inactive alone, fold into functional forms and sPecifically bind tRNA even in the absence of Proteins. Based on the crosslinking results and crystal structures of bacterial RNAs, we develoP a tertiary structure model of the eukaryal RNase P RNA. The eukaryal RNA contains a core structure similar to the bacterial RNA but lacks sPecific features that in bacterial RNAs contribute to catalysis and global stability of tertiary structure.
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Crystal structure of a bacterial Ribonuclease P RNA.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Alexei V. Kazantsev, Angelika A. Krivenko, Daniel J. Harrington, Stephen R. Holbrook, Paul D. Adams, Norman R. PaceAbstract:The x-ray crystal structure of a 417-nt Ribonuclease P RNA from Bacillus stearothermoPhilus was solved to 3.3-A resolution. This RNA enzyme is constructed from a number of coaxially stacked helical domains joined together by local and long-range interactions. These helical domains are arranged to form a remarkably flat surface, which is imPlicated by a wealth of biochemical data in the binding and cleavage of the Precursors of transfer RNA substrate. Previous Photoaffinity crosslinking data are used to Position the substrate on the crystal structure and to identify the chemically active site of the ribozyme. This site is located in a highly conserved core structure formed by intricately interlaced long-range interactions between interhelical sequences.
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Phylogenetic-comParative analysis of the eukaryal Ribonuclease P RNA.
RNA (New York N.Y.), 2000Co-Authors: Daniel N. Frank, Christian Pitulle, Catherine Adamidi, Marissa A. Ehringer, Norman R. PaceAbstract:Ribonuclease P (RNase P) is the ribonucleoProtein enzyme that cleaves 5'-leader sequences from Precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are caPable of catalyzing Pre-tRNA maturation in vitro in the absence of Proteins. An exPlanation of these functional differences will be assisted by a detailed comParison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain Poorly characterized, comPared to their bacterial and archaeal homologs. Hence, we have taken a Phylogenetic-comParative aPProach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from sPecies of ascomycetous fungi rePresentative of the genera Arxiozyma, ClavisPora, Kluyveromyces, Pichia, Saccharomyces, SaccharomycoPsis, TorulasPora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comParative analysis of these and other sequences refines Previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comParison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.
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ComParative structure analysis of vertebrate Ribonuclease P RNA
Nucleic acids research, 1998Co-Authors: Christian Pitulle, M. Garcia-paris, Kelly R. Zamudio, Norman R. PaceAbstract:Ribonuclease P cleaves 5'-Precursor sequences from Pre-tRNAs. All cellular RNase P holoenzymes contain homologous RNA elements; the eucaryal RNase P RNA, in contrast to the bacterial RNA, is catalytically inactive in the absence of the Protein comPonent(s). To understand the function of eucaryal RNase P RNA, knowledge of its structure is needed. Considerable effort has been devoted to comParative studies of the structure of this RNA from diverse organisms, including eucaryotes, Primarily fungi, but also a limited set of vertebrates. The substantial differences in the sequences and structures of the vertebrate RNAs from those of other organisms have made it difficult to align the vertebrate sequences, thus limiting comParative studies. To exPand our understanding of the structure of diverse RNase P RNAs, we have isolated by PCR and sequenced 13 Partial RNase P RNA genes from 11 additional vertebrate taxa rePresenting most extant major vertebrate lineages. Based on a recently ProPosed structure of the core elements of RNase P RNA, we aligned the sequences and ProPose a minimum consensus secondary structure for the vertebrate RNase P RNA.
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Ribonuclease P: Unity and Diversity in a tRNA Processing Ribozyme
Annual review of biochemistry, 1998Co-Authors: Daniel N. Frank, Norman R. PaceAbstract:Ribonuclease P (RNase P) is the endoRibonuclease that generates the mature 5'-ends of tRNA by removal of the 5'-leader elements of Precursor-tRNAs. This enzyme has been characterized from rePresentatives of all three domains of life (Archaea, Bacteria, and Eucarya) (1) as well as from mitochondria and chloroPlasts. The cellular and mitochondrial RNase Ps are ribonucleoProteins, whereas the most extensively studied chloroPlast RNase P (from sPinach) is comPosed solely of Protein. Remarkably, the RNA subunit of bacterial RNase P is catalytically active in vitro in the absence of the Protein subunit (2). Although RNA-only activity has not been demonstrated for the archael, eucaryal, or mitochondrial RNAs, comParative sequence analysis has established that these RNAs are homologous (of common ancestry) to bacterial RNA. RNase P holoenzymes vary greatly in organizational comPlexity across the Phylogenetic domains, Primarily because of differences in the RNase P Protein subunits: Mitochondrial, archaeal, and eucaryal holoenzymes contain larger, and PerhaPs more numerous, Protein subunits than do the bacterial holoenzymes. However, that the nonbacterial RNase P RNAs retain significant structural similarity to their catalytically active bacterial counterParts indicates that the RNA remains the catalytic center of the enzyme.
Denis Drainas - One of the best experts on this subject based on the ideXlab platform.
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PreParation of sPermine conjugates with acidic retinoids with Potent Ribonuclease P inhibitory activity.
European journal of medicinal chemistry, 2009Co-Authors: George D. Magoulas, Evangelia Papadimou, Dionysios Papaioannou, Denis DrainasAbstract:Novel mono- and diacylated sPermines, readily obtained using isolable succinimidyl active esters of acidic retinoids for the selective acylation of free sPermine or in situ activated acidic retinoids for acylating selectively Protected sPermine followed by deProtection, were shown to inhibit the ribozyme Ribonuclease P more strongly than the Parent retinoids.
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Activation of bacterial Ribonuclease P by macrolides.
Biochemistry, 2008Co-Authors: Chrisavgi Toumpeki, Anastassios Vourekas, Dimitra Kalavrizioti, Vasiliki Stamatopoulou, Denis DrainasAbstract:The effect of macrolide antibiotic sPiramycin on RNase P holoenzyme and M1 RNA from Escherichia coli was investigated. Ribonuclease P (RNase P) is a ribozyme that is resPonsible for the maturation of 5' termini of tRNA molecules. SPiramycin revealed a dose-dePendent activation on Pre-tRNA cleavage by E. coli RNase P holoenzyme and M1 RNA. The K s and V max, as well as the K s(aPP) and V max(aPP) values of RNase P holoenzyme and M1 RNA in the Presence or absence of sPiramycin, were calculated from Primary and secondary kinetic Plots. It was found that the activity status of RNase P holoenzyme and M1 RNA is imProved by the Presence of sPiramycin 18- and 12-fold, resPectively. Primer extension analysis revealed that sPiramycin induces a conformational change of the P10/11 structural element of M1 RNA, which is involved in substrate recognition.
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DRPP20 and DRPP40: Two Protein subunits involved in Dictyostelium discoideum Ribonuclease P holoenzyme assembly.
Gene, 2007Co-Authors: Dimitra Kalavrizioti, Anastassios Vourekas, Denis DrainasAbstract:Ribonuclease P is an essential enzyme that matures the 5' ends of all Primary tRNA transcriPts. RNase P enzymes contain a similar in size RNA subunit which is absolutely required for catalysis. The holoenzyme from Dictyostelium discoideum Possesses an essential for activity RNA subunit but the exact Protein comPosition is still under investigation. Bioinformatic analysis of D. discoideum sequencing data returned seven ORFs homologous to Previously characterized RNase P Protein subunits from human. In the Present study, DRPP20 and DRPP40 were cloned and characterized. These Proteins aPart from the noted similarity Possess idiosyncratic regions. Immunobiochemical analysis Presented herein indicates their direct involvement in the formation of the ribonucleoProtein comPlex of D. discoideum RNase P holoenzyme.
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Inhibition of eukaryotic Ribonuclease P activity by aminoglycosides: kinetic studies.
FEBS letters, 2000Co-Authors: Apostolos Tekos, Antigoni Tsagla, Constantinos Stathopoulos, Denis DrainasAbstract:The effect of several aminoglycoside antibiotics on Ribonuclease P (RNase P) was investigated using an in vitro exPerimental system from Dictyostelium discoideum. Detailed kinetic analysis showed that all aminoglycosides tested (tobramycin, gentamicin, kanamycin, Paromomycin, neomycin) behave as classical non-comPetitive inhibitors, with neomycin being the strongest inhibitor. The inhibition effect is attributed to the electrostatic comPetition of the cationic aminoglycosides with magnesium ions required for catalysis. Increasing Mg2+ ion concentrations reduced the effect of aminoglycosides on RNase P activity. Detailed kinetic analysis showed that aminoglycosides comPete with Mg2+ for common binding sites on RNase P holoenzyme.
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Dose-dePendent inhibition of Ribonuclease P activity by anthralin.
Skin pharmacology and applied skin physiology, 2000Co-Authors: Denis Drainas, Evangelia Papadimou, Alexandra Monastirli, Dionysios Tsambaos, Hans F. MerkAbstract:The effect of five different anthralin concentrations on tRNA biogenesis was investigated emPloying the Ribonuclease P (RNase P) of the slime mold Dictyostelium discoideum as an in vitro cell-free exPerimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA Precursors to Produce the mature 5' end. Anthralin revealed a dose-dePendent inhibition of RNase P activity indicating that this comPound may have a direct effect on tRNA biogenesis. Taking into account that anthralin has no structural similarities to the substrate (Pre-tRNA) of RNase P, it seems reasonable to suggest that this comPound may bind to allosteric inhibition sites of the enzyme.
