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Allen W Nicholson - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation
Scientific Reports, 2016Co-Authors: Swapna Gone, Mercedes Alfonso-prieto, Samridhdi Paudyal, Allen W NicholsonAbstract:Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli ( Ec ) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec -RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in k_cat and a 1.4-fold decrease in K_m following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.
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Combined computational and experimental analysis of a complex of Ribonuclease III and the regulatory macrodomain protein, YmdB
Proteins, 2015Co-Authors: Samridhdi Paudyal, Mercedes Alfonso-prieto, Shiv K. Redhu, Vincenzo Carnevale, Michael L. Klein, Allen W NicholsonAbstract:Ribonuclease III is a conserved bacterial endonuclease that cleaves double-stranded(ds) structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control that in turn confer global post-transcriptional regulation. The Escherichia coli macrodomain protein YmdB directly interacts with RNase III, and an increase in YmdB amount in vivo correlates with a reduction in RNase III activity. Here, a computational-based structural analysis was performed to identify atomic-level features of the YmdB-RNase III interaction. The docking of monomeric E. coli YmdB with a homology model of the E. coli RNase III homodimer yields a complex that exhibits an interaction of the conserved YmdB residue R40 with specific RNase III residues at the subunit interface. Surface Plasmon Resonance (SPR) analysis provided a KD of 61 nM for the complex, corresponding to a binding free energy (ΔG) of −9.9 kcal/mol. YmdB R40 and RNase III D128 were identified by in silico alanine mutagenesis as thermodynamically important interacting partners. Consistent with the prediction, the YmdB R40A mutation causes a 16-fold increase in KD (ΔΔG = +1.8 kcal/mol), as measured by SPR, and the D128A mutation in both RNase III subunits (D128A/D128′A) causes an 83-fold increase in KD (ΔΔG = +2.7 kcal/mol). The greater effect of the D128A/D128′A mutation may reflect an altered RNase III secondary structure, as revealed by CD spectroscopy, which also may explain the significant reduction in catalytic activity in vitro. The features of the modeled complex relevant to potential RNase III regulatory mechanisms are discussed. Proteins 2015; 83:459–472. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
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Interaction of Ribonuclease III with the Regulatory Macrodomain Protein YmdB Analyzed by Docking Calculations and SPR Experiments
Biophysical Journal, 2015Co-Authors: Mercedes Alfonso-prieto, Samridhdi Paudyal, Shiv K. Redhu, Vincenzo Carnevale, Michael L. Klein, Allen W NicholsonAbstract:Ribonuclease III (RNase III) is a conserved bacterial endonuclease that cleaves double-stranded(ds) RNA structures and is essential in diverse RNA maturation and decay pathways [1,2]. RNase III is subject to multiple levels of regulation, allowing fine-tuning of its catalytic activity depending on the cellular physiological state. The regulatory macrodomain protein YmdB interacts with RNase III, and an increase in YmdB levels correlates with a decrease in RNase III activity in vivo [3]. However, the molecular details of the YmdB-RNase III interaction are not yet known. Here, docking calculations and computationally-driven mutagenesis were combined with surface plasmon resonance (SPR) experiments to identify energetically important determinants of the Escherichia coli YmdB-RNase III interaction. The computational results reveal two alternative YmdB binding sites in RNase III: one located in the N-terminal nuclease domain (RIIID) (also indicated by co-immunoprecipitation and chemical cross-linking [3]), and a novel site in the C-terminal dsRNA-binding domain (dsRBD). The binding site in the RIIID is composed of a cluster of negatively charged residues that interact with a conserved arginine in YmdB, and the importance of this interaction is confirmed by SPR analysis of the YmdB Arg to Ala mutation. These results suggest a mechanism of RNase III regulation in which YmdB can bind separate sites in a concentration-dependent manner, leading to inhibition of catalytic activity.[1] Nicholson A.W. 2014. Ribonuclease III mechanisms of double-stranded RNA cleavage. WIREs RNA 5:31-48.[2] Court D.L., Gan J., Liang Y.-H., Shaw G.X., Tropea J.E., Costantino N., Waugh D.S., Ji X. 2013. RNase III: genetics and function; structure and mechanism. Annu. Rev. Genet. 47:405-431.[3] Kim K.-S., Manasherob R., Cohen S.N. 2008. YmdB: a stress-responsive Ribonuclease-binding regulator of E. coli RNase III activity. Genes Dev. 22:3497-3508.
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Ribonuclease III mechanisms of double stranded rna cleavage
Wiley Interdisciplinary Reviews - Rna, 2014Co-Authors: Allen W NicholsonAbstract:Double-stranded(ds) RNA has diverse roles in gene expression and regulation, host defense, and genome surveillance in bacterial and eukaryotic cells. A central aspect of dsRNA function is its selective recognition and cleavage by members of the Ribonuclease III (RNase III) family of divalent-metal-ion-dependent phosphodiesterases. The processing of dsRNA by RNase III family members is an essential step in the maturation and decay of coding and noncoding RNAs, including miRNAs and siRNAs. RNase III, as first purified from Escherichia coli, has served as a biochemically well-characterized prototype, and other bacterial orthologs provided the first structural information. RNase III family members share a unique fold (RNase III domain) that can dimerize to form a structure that binds dsRNA and cleaves phosphodiesters on each strand, providing the characteristic 2 nt, 3′-overhang product ends. Ongoing studies are uncovering the functions of additional domains, including, inter alia, the dsRNA-binding and PAZ domains that cooperate with the RNase III domain to select target sites, regulate activity, confer processivity, and support the recognition of structurally diverse substrates. RNase III enzymes function in multicomponent assemblies that are regulated by diverse inputs, and at least one RNase III-related polypeptide can function as a noncatalytic, dsRNA-binding protein. This review summarizes the current knowledge of the mechanisms of catalysis and target site selection of RNase III family members, and also addresses less well understood aspects of these enzymes and their interactions with dsRNA. WIREs RNA 2014, 5:31–48. doi: 10.1002/wrna.1195
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Ribonuclease III mechanisms of double-stranded RNA cleavage.
Wiley interdisciplinary reviews. RNA, 2013Co-Authors: Allen W NicholsonAbstract:Double-stranded(ds) RNA is created through the pairing of complementary sequences. A ubiquitous motif in biological systems, dsRNA exhibits a limited conformational plasticity, but exerts a multitude of biological effects. dsRNA can possess a transient, dynamic nature, as seen in antisense RNA binding to target sites. Intramolecular base-pairing provides local double-helical structures that can function as architectural elements or as protein recognition sites. In the latter case, protein binding to dsRNA enables processes such as RNA transport and localization. dsRNA invokes potent cellular responses, as a single molecule of dsRNA can trigger interferon production in vertebrate cells. The cellular response underscores the important aspect of dsRNA as an indicator of pathogenic conditions. In fact, dsRNA is the chromosomal material of many viruses, and cell membrane receptors specifically recognize dsRNA as an initial event in the response pathway.1–5 The enzymatic cleavage of dsRNA is a conserved reaction of fundamental importance in cellular and viral gene expression and regulation, host defense, and genome surveillance. A conserved enzymatic mechanism has been characterized that catalyzes the coordinate cleavage of both RNA strands at selected target sites. Specifically, dsRNA processing is accomplished by members of a Ribonuclease family, including the prototypical Ribonuclease III (RNase III) of bacterial cells. Ongoing studies on RNase III, and in particular the enzyme from Escherichia coli, have revealed a global role for dsRNA processing in bacterial gene expression and regulation.6,7 The subsequent characterization of eukaryotic family members has extended the essential involvement of dsRNA processing in many additional pathways.8–10 In particular, analysis of the components of RNA interference (RNAi) and related pathways identified two RNase III family members, Dicer and Drosha, as essential functional partners in RNAi.10 Understanding how dsRNA is processed by RNase III family members, and characterizing the many consequences of this reaction remain as key objectives. This review addresses current knowledge of dsRNA processing by RNase III family enzymes. Emphasis will be placed on the mechanisms of catalysis and target site selection. The salient physicochemical properties of dsRNA also will be summarized. Owing to page limitations this review is unable to include the important contributions from many laboratories that are studying dsRNA processing and gene regulation. However, the reviews cited above may serve as alternative sources.
James L. Van Etten - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a chlorella virus PBCV-1 encoded Ribonuclease III ☆
Virology, 2003Co-Authors: Yuanzheng Zhang, Allen W Nicholson, Irina E. Calin-jageman, James R. Gurnon, Tae-jin Choi, Byron J. Adams, James L. Van EttenAbstract:Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30 –35% amino acid identity to Ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in aM g 2 -dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5–10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniaeRNase III.
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characterization of a chlorella virus pbcv 1 encoded Ribonuclease III
Virology, 2003Co-Authors: Yuanzheng Zhang, Allen W Nicholson, Irina E Calinjageman, James R. Gurnon, Tae-jin Choi, Byron J. Adams, James L. Van EttenAbstract:Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30 –35% amino acid identity to Ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in aM g 2 -dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5–10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniaeRNase III.
Irina E. Calin-jageman - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a chlorella virus PBCV-1 encoded Ribonuclease III ☆
Virology, 2003Co-Authors: Yuanzheng Zhang, Allen W Nicholson, Irina E. Calin-jageman, James R. Gurnon, Tae-jin Choi, Byron J. Adams, James L. Van EttenAbstract:Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30 –35% amino acid identity to Ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in aM g 2 -dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5–10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniaeRNase III.
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Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli Ribonuclease III substrates.
Biochemistry, 2003Co-Authors: Irina E. Calin-jageman, Allen W NicholsonAbstract:The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs. The primary agents of dsRNA processing are members of the Ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells. Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs. E. coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity. RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes. Some E. coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester. The specific features of the internal loop that establish the pattern of single-strand cleavage are not known. A mutational analysis of the asymmetric [4 nt/5 nt] inte...
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RNA structure‐dependent uncoupling of substrate recognition and cleavage by Escherichia coli Ribonuclease III
Nucleic acids research, 2003Co-Authors: Irina E. Calin-jageman, Allen W NicholsonAbstract:Members of the Ribonuclease III superfamily of double-strand-specific endoRibonucleases participate in diverse RNA maturation and decay pathways. Ribonuclease III of the gram-negative bacterium Escherichia coli processes rRNA and mRNA precursors, and its catalytic action can regulate gene expression by controlling mRNA translation and stability. It has been proposed that E.coli RNase III can function in a non-catalytic manner, by binding RNA without cleaving phosphodiesters. However, there has been no direct evidence for this mode of action. We describe here an RNA, derived from the T7 phage R1.1 RNase III substrate, that is resistant to cleavage in vitro by E.coli RNase III but retains comparable binding affinity. R1.1[CL3B] RNA is recognized by RNase III in the same manner as R1.1 RNA, as revealed by the similar inhibitory effects of a specific mutation in both substrates. Structure-probing assays and Mfold analysis indicate that R1.1[CL3B] RNA possesses a bulge– helix–bulge motif in place of the R1.1 asymmetric internal loop. The presence of both bulges is required for uncoupling. The bulge–helix–bulge motif acts as a ‘catalytic’ antideterminant, which is distinct from recognition antideterminants, which inhibit RNase III binding.
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Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli Ribonuclease III.
Nucleic acids research, 2001Co-Authors: Irina E. Calin-jageman, Asoka K. Amarasinghe, Allen W NicholsonAbstract:Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli Ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (III) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.
Rhonda H. Nicholson - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Aquifex aeolicus Ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates
Nucleic acids research, 2010Co-Authors: Zhongjie Shi, Rhonda H. Nicholson, Ritu Jaggi, Allen W NicholsonAbstract:Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ∼70–85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III.
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New approaches to understanding double-stranded RNA processing by Ribonuclease III purification and assays of homodimeric and heterodimeric forms of RNase III from bacterial extremophiles and mesophiles.
Methods in enzymology, 2008Co-Authors: Wenzhao Meng, Lilian Nathania, Rhonda H. Nicholson, Alexandre V. Pertzev, Allen W NicholsonAbstract:Abstract Ribonuclease III (RNase III) is a double‐stranded (ds)‐RNA–specific endonuclease that plays essential roles in the maturation and decay of coding and noncoding RNAs. Bacterial RNases III are structurally the simplest members of the RNase III family, which includes the eukaryotic orthologs Dicer and Drosha. High‐resolution crystal structures of RNase III of the hyperthermophilic bacteria Aquifex aeolicus and Thermotoga maritima are available. A. aeolicus RNase III also has been cocrystallized with dsRNA or specific hairpin substrates. These structures have provided essential structural insight to the mechanism of dsRNA recognition and cleavage. However, comparatively little is known about the catalytic behaviors of A. aeolicus or T. maritima RNases III. This chapter provides protocols for the purification of A. aeolicus and T. maritima RNases III and also describes the preparation of artificial heterodimers of Escherichia coli RNase III, which are providing new insight on the subunit and domain interactions involved in dsRNA recognition and cleavage.
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Mouse Ribonuclease III. cDNA structure, expression analysis, and chromosomal location.
BMC genomics, 2002Co-Authors: Kristine R Fortin, Rhonda H. Nicholson, Allen W NicholsonAbstract:Background Members of the Ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoRibonucleases participate in diverse RNA maturation and decay pathways in eukaryotic and prokaryotic cells. A human RNase III orthologue has been implicated in ribosomal RNA maturation. To better understand the structure and mechanism of mammalian RNase III and its involvement in RNA metabolism we determined the cDNA structure, chromosomal location, and expression patterns of mouse RNase III.
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molecular characterization of a mouse cdna encoding dicer a Ribonuclease III ortholog involved in rna interference
Mammalian Genome, 2002Co-Authors: Rhonda H. Nicholson, Allen W NicholsonAbstract:Members of the Ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoRibonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named "Dicer", functions in the RNA interference (RNAi) pathway by producing 21--23 bp dsRNAs which target the selective destruction of homologous RNAs. RNAi is operative in animals, plants, and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as to participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer ortholog. On the basis of the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences; and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse Chromosome (Chr) 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III ortholog responsible for dsRNA processing in the RNAi pathway.
Yuanzheng Zhang - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a chlorella virus PBCV-1 encoded Ribonuclease III ☆
Virology, 2003Co-Authors: Yuanzheng Zhang, Allen W Nicholson, Irina E. Calin-jageman, James R. Gurnon, Tae-jin Choi, Byron J. Adams, James L. Van EttenAbstract:Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30 –35% amino acid identity to Ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in aM g 2 -dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5–10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniaeRNase III.
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characterization of a chlorella virus pbcv 1 encoded Ribonuclease III
Virology, 2003Co-Authors: Yuanzheng Zhang, Allen W Nicholson, Irina E Calinjageman, James R. Gurnon, Tae-jin Choi, Byron J. Adams, James L. Van EttenAbstract:Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30 –35% amino acid identity to Ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in aM g 2 -dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5–10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniaeRNase III.